Brachyury polypeptides and methods for use

ABSTRACT

It is disclosed herein that Brachyury is expressed in human tumors, specifically in tumors of the small intestine, stomach, kidney, bladder, uterus, ovary, and testes, as well as in lung, colon and prostate carcinomas. Immunogenic Brachyury polypeptides are disclosed herein. These polypeptides can be used in diagnostic assays for Brachyury expression, as well as for inducing an immune response to Brachyury. Polynucleotides encoding the immunogenic Brachyury polypeptides, vectors including these polypeptides, host cells transformed with these vectors, and methods of using these polypeptides, polynucleotides, vectors, and host cells are provided. Methods of diagnosing a Brachyury-expressing cancer are also provided. Exemplary cancers include small lung, colon, intestine, stomach, kidney, bladder, uterus, ovary, and testes and prostate cancers. Methods of treating cancer are also disclosed.

PRIORITY CLAIM

This is a continuation of U.S. patent application Ser. No. 12/528,796,filed Aug. 26, 2009, which is the U.S. national stage of PCT ApplicationNo. PCT/US2008/055185, filed Feb. 27, 2008, which was published inEnglish under PCT Article 21(2), which in turn claims the benefit ofU.S. Provisional Application No. 60/904,236, filed Feb. 28, 2007. All ofthe prior applications are incorporated herein by reference in theirentirety.

FIELD

This application relates to the field of cancer therapeutics,specifically to molecules such as immunogenic peptides and inhibitorynucleic acids for the treatment of cancer.

BACKGROUND

Brachyury (also known as “T”) was identified in mice as a dominant shorttail mutant that is also a recessive lethal; homozygous T/T embryos diein mid-gestation due to a failure of posterior mesoderm formation(Chesley, J. Exp. Zool., 70: 429-459, 1935). The murine Brachyury genehas been cloned (Herrmann et al., Nature (Lond.), 343: 617-622, 1990),as well as the homologs in other species, such as humans. The expressionof the human homologue of the mouse Brachyury was detected by RT-PCR inthe notochord remnant, the nucleus pulposus, of human abortuses at 14-15weeks gestation (Edwards et al., Genome Res., 6: 226-233, 1996).

Brachyury has generally proved a valuable marker for recognition ofmesodennal differentiation (Herrmann et al., Trends Genet., 10: 280-286,1994). For example, apart from expression in embryos themselves,Brachyury has been reported to be activated during the differentiationof certain murine EC and ES cell lines differentiating along mesodennallineages in vitro (see, for example, Bain et al., Biochem. Biophys. Res.Commun., 223: 691-694, 1996). In humans, Brachyury has been shown to beexpressed in teratocarcinomas (Gokhele et al., Cell Growth andDifferentiation 11:157-62, 2000), chordomas (Vujovic et al., J. Pathol.2: 157-65, 2006) and hemagioblastomas (Glasker et al., Cancer Res. 66:4167-4172, 2006).

Immunotherapy involves evoking an immune response against cancer cellsbased on their production of target antigens. Immunotherapy based oncell-mediated immune responses involves generating a cell-mediatedresponse to cells that produce particular antigenic determinants, whileimmunotherapy based on humoral immune responses involves generatingspecific antibodies to cells that produce particular antigenicdeterminants.

Recent studies show that immunotherapy of cancer patients may bedramatically improved by the finding that CD8⁺ CTLs recognize and killtumor cells that display peptides from tumor-associated antigens withinMHC Class I molecules. In clinical studies it has been found thateffector CD8⁺ T cells play a major role in tumor regression. Forexample, several tumor antigens in prostate cancer models have beenidentified and HLA allele-specific peptides from those prostatecancer-associated antigens have been identified as CD8⁺ T cell epitopes.For example, HLA-A2.1 binding peptides were described that were derivedfrom prostate specific antigen (PSA) (Correale et al., J Immunol161:3186, 1998), prostate-specific membrane antigen (PSMA) (Tjoa et al.,Prostate 28:65, 1996), prostate stem cell antigen (PSCA) (Kiessling etal., Int J Cancer 102:390, 2002), and prostate acid phosphatase (Peshwaet al., Prostate 36:129, 1998). For PSA, clinical trials are in progressusing different vaccine strategies. However, there clearly is a need toidentify additional antigens to aid in the diagnosis of cancers ofdifferent organs, and to produce peptides that can be used forimmunotherapy of other types of cancer.

SUMMARY

It is disclosed herein that Brachyury is expressed in human tumors,specifically in tumors of the small intestine, stomach, kidney, bladder,uterus, ovary, and testes, as well as in lung, colon and prostatecarcinomas. Immunogenic Brachyury polypeptides are disclosed herein.These Brachyury polypeptides can be used for inducing an immune responseto Brachyury, as well as in diagnostic assays for Brachyury expression.In one example, the polypeptide is at most twelve consecutive aminoacids in length, wherein the isolated polypeptide comprises the aminoacid sequence set forth as WLLPGTSTX₁ (SEQ ID NO: 3), wherein X₁ is aleucine (L) or a valine (V).

Polynucleotides encoding the immunogenic Brachyury polypeptides, vectorsincluding these polypeptides, host cells transformed with these vectors,and methods of using these polypeptides, polynucleotides, vectors, andhost cells are provided herein. In one embodiment, a composition isdisclosed that includes a first recombinant virus which has incorporatedinto a viral genome or infectable portion thereof a nucleic acidencoding the immunogenic Brachyury polypeptide and a second recombinantvirus which has incorporated into a viral genome or infectable portionthereof one or more genes or DNA sequences encoding B7-1, B7-2, or B7-1and B7-2, wherein the composition is able to coinfect a host cellresulting in co-expression of the polypeptide and the B7-1, B7-2, orB7-1 and B7-2 encoding genes or DNA sequences.

Methods of diagnosing a Brachyury-expressing cancer are also provided,that include the use of the disclosed immunogenic Brachyurypolypeptides, nucleic acids encoding these polypeptides, or antibodiesthat specifically bind these polypeptides. Exemplary cancers includelung, colon, small intestine, stomach, kidney, bladder, uterus, ovary,and testes and prostate cancers.

Methods of inducing an immune response to Brachyury are also disclosed.The methods include the use of the immunogenic Brachyury polypeptidesdisclosed herein, nucleic acids encode these polypeptides, and/or viralvectors encoding an immunogenic Brachyury polypeptide, alone or inconjunction with other agents, such as B7-1, B7-2, and/or a cytokineand/or with traditional cancer therapies, such as surgery, radiationtherapy and/or chemotherapy. Methods are disclosed for treating asubject having a tumor, such as, but not limited to, a small intestine,stomach, kidney bladder, uterus, ovary, testes, lung, colon or prostatetumor. Methods are also disclosed for treating a subject having a breasttumor, bronchial tube tumor, chronic lymphocytic leukemia (CLL) andother B cell-based malignancies. These methods include inducing animmune response to Brachyury and/or using an inhibitory nucleic acid,such as an siRNA or antisense molecule, to decrease Brachyury expressionin order to treat the tumor.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C are a set of digital images of RT-PCR analysis of Brachyuryexpression in human normal and tumor tissues. FIG. 1A is a digital imageof RT-PCR results from human multiple tissues cDNA panels I and II. FIG.1B is a digital image of RT-PCR results from various human bloodfraction cDNAs. FIG. 1C is a digital image of results from RT-PCR ofcDNA from tumor tissues (each tissue from an individual cancer patient)that were amplified for expression of Brachyury (upper panel) and GAPDH(bottom panel). Human DNA was used as a positive control for the PCRreaction; water was added to the tubes labeled as negative control.

FIGS. 2A-2B are graphs illustrating the binding of predicted peptides toHLA-A0201 molecules. FIG. 2A is a bar graph of results wherein peptidesat 25 μM were analyzed for binding to T2 cells; a positive control(CAP1-6D) and an HLA-A3 binding peptide (negative control) were used atthe same concentration. MFI indicates mean fluorescence intensity. FIG.2B is a line graph of results showing an analysis of half-life ofpeptide-MHC complexes that was conducted as described in Materials andMethods. For each peptide and the positive control CAP1-6D, half-lifetime is given in minutes.

FIG. 3A-3C are graphs showing cytokine production and cytotoxic activityof CTLs specific for three Brachyury derived peptides. FIG. 3A is a bargraph showing results obtained when CD8+ T cells generated from PBMC ofa healthy donor against peptides T-p2, T-p3, and T-p4 were stimulatedfor 24 hours in the presence of Brachyury (T)-specific peptides orirrelevant peptide-pulsed autologous DCs. IFN-γ was evaluated in thesupernatants by ELISA. FIG. 3B is a line graph showing cytotoxicactivity (6-hour assay) of CTLs generated with peptides T-p2 and T-p3against peptide-pulsed C1R-A2 targets. Two effector-to-target ratios(E:T) were used as indicated. C1R-A2 cells were pulsed with 25 μM ofT-p2 peptide (closed circles), T-p3 peptide (open circles), irrelevantCAP1-6D peptide (close triangles), and without peptide (open triangles).FIG. 3C is a line graph of results obtained when T2 cells were pulsedwith various concentrations of T-p2 peptide as indicated and used astargets with T-p2 CTLs (at an effector-to-targets ratio equal to12.5:1).

FIGS. 4A-4F are graphs showing the Cytotoxic activity ofBrachyury-specific CTLs against tumor targets. FIG. 4A is a line graphof results obtained when T-p2 CTLs from a normal donor were used aseffectors against various tumor targets in an ¹¹¹In 16-hour releaseassay, as indicated. FIG. 4B is a bar graph of results obtained when¹¹¹In-labeled H441 tumor cells were incubated with 25 μg/ml of a controlIgG, anti-HLA-class I, or anti-HLA-class II MAb for 1 hour before theaddition of T-p2 T cells. The E:T ratio was 20:1. FIG. 4C is a linegraph of results obtained when CTLs established from the blood of acolorectal cancer patient (patient 1) and (FIG. 4D) an ovarian cancerpatient (patient 2) were used after three IVS for cytotoxic killing ofH441 and AsPC-1 tumor cells. FIG. 4E is a bar graph showing cytotoxickilling of LNCAP tumor cells by T-p2 T cells derived from patient 1.¹¹¹In-labeled LNCAP tumor cells were incubated with 25 μg/ml of acontrol IgG or an anti-HLA-A2,28 MAb for 1 hour before the addition ofT-p2 T cells. FIG. 4F is a bar graph and a digital image of resultsobtained when T cells derived from patient 2 were used as effectorsagainst various tumor targets, as indicated. Shown in the digital imageis the expression of Brachyury and β-actin mRNA by RT-PCR in each tumorcell line.

FIGS. 5A-5B are digital images showing that stable knockdown ofBrachyury expression induces a mesenchymal-to-epithelial transition inNCI-H460 lung carcinoma cancer cells. FIG. 5A is a digital image of anRT-PCR analysis of Brachyury and β-actin mRNA expression in NCI-H460lung carcinoma cells stably transfected with a control shRNA or aBrachyury-specific shRNA construct (Br.shRNA clones 1 and 2). FIG. 5B isa digital image of the results obtained when the same cell lines wereanalyzed by western blot for expression of human fibronectin, vimentin,γ-catenin, and β-actin.

FIGS. 6A-6B are digital images and bar graphs showing that the loss ofBrachyury impairs the migratory and invasive properties of NCI-H460 lungcarcinoma cells in vitro. In vitro assay of (FIG. 6A) cell migration and(FIG. 6B) cell invasion in NCI-H460 lung carcinoma cells stablytransfected with a control shRNA or a Brachyury-specific shRNA construct(Br.shRNA clone 2). Experiments (n=3) were conducted in triplicatesamples of each cell line as described in the Examples section. Thegraph shows results from one representative experiment. Each barrepresents the results for an individual replicate assay±SEM.Representative images of the bottom side of the filters for each cellline under ×10 magnification are also shown. Statistical analysis ofStudent's t-test was performed.

SEQUENCE LISTING

The Sequence Listing is submitted as an ASCII text file[4239-77527-10_Sequence_Listing.txt, Apr. 25, 2012, 11.6 KB], which isincorporated by reference herein.

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 is an exemplary amino acid sequence for a Brachyuryprotein.

SEQ ID NO: 2 is an exemplary nucleic acid sequence encoding a Brachyurypolypeptide.

SEQ ID NO: 3 is the amino acid sequence of an immunogenic Brachyurypolypeptide.

SEQ ID NOS: 4-11 are the nucleic acid sequences of primers.

SEQ ID NO: 12 is the amino acid sequence of a carcinoembryonic antigen(CEA) polypeptide.

SEQ ID NO: 13 is the amino acid sequence of a human immunodeficiencyvirus (HIV) polypeptide.

SEQ ID NOS: 14-22 are the amino acid sequence of exemplary Brachyurypolypeptides.

DETAILED DESCRIPTION

It is disclosed herein that Brachyury is expressed in human tumors,specifically in tumors of the small intestine, stomach, kidney, bladder,uterus, ovary, and testes, as well as in lung, colon and prostatecarcinomas. Brachyury is also expressed in chronic lymphocytic leukemiaand other B cell malignancies. Immunogenic Brachyury polypeptides aredisclosed herein. Nucleic acids encoding these polypeptides, vectorsincluding these nucleic acids, and host cells transformed with thevectors are also disclosed. Methods for inducing an immune response to atumor cell expressing Brachyury are also disclosed, as are methods fordetecting a tumor that expresses Brachyury. Methods for treatment arealso disclosed herein for the treatment of a tumor that expressesBrachyury, wherein the method includes administering an inhibitorynucleic acid.

TERMS

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common teens in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided,along with particular examples:

Adjuvant: A vehicle used to enhance antigenicity. Adjuvants include asuspension of minerals (alum, aluminum hydroxide, or phosphate) on whichantigen is adsorbed; or water-in-oil emulsion in which antigen solutionis emulsified in mineral oil (Freund incomplete adjuvant), sometimeswith the inclusion of killed mycobacteria (Freund's complete adjuvant)to further enhance antigenicity (inhibits degradation of antigen and/orcauses influx of macrophages). Immunstimulatory oligonucleotides (suchas those including a CpG motif) can also be used as adjuvants (forexample see U.S. Pat. No. 6,194,388; U.S. Pat. No. 6,207,646; U.S. Pat.No. 6,214,806; U.S. Pat. No. 6,218,371; U.S. Pat. No. 6,239,116; U.S.Pat. No. 6,339,068; U.S. Pat. No. 6,406,705; and U.S. Pat. No.6,429,199). Adjuvants include biological molecules (a “biologicaladjuvant”), such as costimulatory molecules. Exemplary adjuvants includeIL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2,OX-40L and 41 BBL.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes. “Epitope” or “antigenicdeterminant” refers to a site on an antigen to which B and/or T cellsrespond. In one embodiment, T cells respond to the epitope, when theepitope is presented in conjunction with an MHC molecule. Epitopes canbe formed both from contiguous amino acids or noncontiguous amino acidsjuxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, and more usually, at least 5, about 9, or about 8-10 aminoacids in a unique spatial conformation. Methods of determining spatialconformation of epitopes include, for example, x-ray crystallography and2-dimensional nuclear magnetic resonance.

An antigen can be a tissue-specific antigen, or a disease-specificantigen. These terms are not exclusive, as a tissue-specific antigen canalso be a disease specific antigen. A tissue-specific antigen isexpressed in a limited number of tissues, such as a single tissue.Specific, non-limiting examples of a tissue specific antigen are aprostate specific antigen, a uterine specific antigen, and/or a testesspecific antigen. A tissue specific antigen may be expressed by morethan one tissue, such as, but not limited to, an antigen that isexpressed in more than one reproductive tissue, such as in both prostateand uterine tissue. A disease-specific antigen is expressedcoincidentally with a disease process. Specific non-limiting examples ofa disease-specific antigen are an antigen whose expression correlateswith, or is predictive of, tumor formation, such as prostate cancerand/or uterine cancer and/or testicular cancer. A disease-specificantigen can be an antigen recognized by T cells or B cells.

Amplification: Of a nucleic acid molecule (e.g., a DNA or RNA molecule)refers to use of a technique that increases the number of copies of anucleic acid molecule in a specimen. An example of amplification is thepolymerase chain reaction, in which a biological sample collected from asubject is contacted with a pair of oligonucleotide primers, underconditions that allow for the hybridization of the primers to a nucleicacid template in the sample. The primers are extended under suitableconditions, dissociated from the template, and then re-annealed,extended, and dissociated to amplify the number of copies of the nucleicacid. The product of amplification can be characterized byelectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing using standard techniques. Other examples of amplificationinclude strand displacement amplification, as disclosed in U.S. Pat. No.5,744,311; transcription-free isothermal amplification, as disclosed inU.S. Pat. No. 6,033,881; repair chain reaction amplification, asdisclosed in WO 90/01069; ligase chain reaction amplification, asdisclosed in EP-A-320 308; gap filling ligase chain reactionamplification, as disclosed in U.S. Pat. No. 5,427,930; and NASBA™ RNAtranscription-free amplification, as disclosed in U.S. Pat. No.6,025,134.

Antibody: Immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site that specifically binds (immunoreacts with) an antigen.

A naturally occurring antibody (e.g., IgG, IgM, IgD) includes fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinterconnected by disulfide bonds. However, it has been shown that theantigen-binding function of an antibody can be performed by fragments ofa naturally occurring antibody. Thus, these antigen-binding fragmentsare also intended to be designated by the term “antibody.” Specific,non-limiting examples of binding fragments encompassed within the termantibody include (i) a Fab fragment consisting of the V_(L), V_(H),C_(L) and C_(HI) domains; (ii) an F_(d) fragment consisting of the V_(H)and C_(HI) domains; (iii) an Fv fragment consisting of the VL and VHdomains of a single arm of an antibody, (iv) a dAb fragment (Ward etal., Nature 341:544-546, 1989) which consists of a V_(H) domain; (v) anisolated complementarity determining region (CDR); and (vi) a F(ab′)₂fragment, a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region.

Immunoglobulins and certain variants thereof are known and many havebeen prepared in recombinant cell culture (e.g., see U.S. Pat. No.4,745,055; U.S. Pat. No. 4,444,487; WO 88/03565; EP 256,654; EP 120,694;EP 125,023; Faoulkner et al., Nature 298:286, 1982; Morrison, J.Immunol. 123:793, 1979; Morrison et al., Ann Rev. Immunol 2:239, 1984).Humanized antibodies and fully human antibodies are also known in theart.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antisense, Sense, and Antigene: Double-stranded DNA (dsDNA) has twostrands, a 5′->3′ strand, referred to as the plus strand, and a 3′->5′strand (the reverse complement), referred to as the minus strand.Because RNA polymerase adds nucleic acids in a 5′->3′ direction, theminus strand of the DNA serves as the template for the RNA duringtranscription. Thus, the RNA formed will have a sequence complementaryto the minus strand and identical to the plus strand (except that U issubstituted for T).

Antisense molecules are molecules that are specifically hybridizable orspecifically complementary to either RNA or the plus strand of DNA.Sense molecules are molecules that are specifically hybridizable orspecifically complementary to the minus strand of DNA. Antigenemolecules are either antisense or sense molecules directed to a dsDNAtarget.

Brachyury: The Brachyury gene is known to be important for thedevelopment of mesoderm during gastrulation. Brachyury is the foundingmember of a family of transcription factors, designated T-boxtranscription factors, characterized by a conserved DNA-binding domain(Papaioannou and Silver, Bioessays 20(1):9-19, 1998), that has anessential role in the formation and organization of mesodeim invertebrates (see, for example, Kispert and Hellmann, Embo J12(8):3211-20, 1993). For example, in Xenopus, Brachyury is anearly-immediate response gene of mesoderm inducers, such as activin orTGF-β, and injection of Brachyury mRNA in embryos is sufficient toinduce ectopic mesoderm development (Smith et al., Cell 67(1):79-87,1991). In addition to the fundamental role of the T-box proteins in thecontrol of developmental processes, several members of this familyappear to be deregulated in cancer. The human Tbx2 gene has beenreported to be amplified in pancreatic cancer cell lines (Mahlamaki etal., Genes Chromosomes Cancer 35(4):353-8, 2002) and over-expressed inBRCA-1- and BRCA-2-mutated breast tumors (Sinclair et al., Cancer Res62(13):3587-9, 2002). Brachyury expression has been previously reportedin human teratocarcinoma lines: a subset of germ cell tumors,teratocarcinomas are embryonal carcinoma cells with competence formesoderm differentiation (Fan et al., Cancer Res 64(15):5132-9, 2004),as well as in chordomas (Vujovic et al, J Pathol 209(2):157-65, 2006).Exemplary human brachyury amino acid and nucleic acid sequences are setforth in GENBANK® Accession No NP_(—)003172 and GENBANK® Accession No.NM_(—)003181, as available on Feb. 23, 2007, incorporated herein byreference

Cancer or Tumor: A malignant neoplasm that has undergone characteristicanaplasia with loss of differentiation, increase rate of growth,invasion of surrounding tissue, and is capable of metastasis. Forexample, prostate cancer is a malignant neoplasm that arises in or fromprostate tissue, ovarian cancer is a malignant neoplasm that arises inor from ovarian tissue, colon cancer is a malignant neoplasm that arisesin or from colon tissue, and lung cancer is a malignant neoplasm thatarises in the lungs. Residual cancer is cancer that remains in a subjectafter any form of treatment given to the subject to reduce or eradicatethe cancer. Metastatic cancer is a cancer at one or more sites in thebody other than the site of origin of the original (primary) cancer fromwhich the metastatic cancer is derived. Cancer includes, but is notlimited to, sarcomas and carcinomas. Prostate cancer is a malignanttumor, generally of glandular origin, of the prostate. Prostate cancersinclude adenocarcinomas and small cell carcinomas.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease an activityor antigenicity of an antigenic epitope of Brachyury. Specific,non-limiting examples of a conservative substitution include thefollowing examples:

Original Residue Conservative Substitutions Al Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; LeuThe term conservative variation also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid, provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide. Non-conservative substitutions are thosethat reduce an activity or antigenicity.

CD4: Cluster of differentiation factor 4, a T cell surface protein thatmediates interaction with the MHC Class II molecule. CD4 also serves asthe primary receptor site for HIV on T cells during HIV infection. Cellsthat express CD4 are often helper T cells.

CD8: Cluster of differentiation factor 8, a T cell surface protein thatmediates interaction with the MHC Class I molecule. Cells that expressCD8 are often cytotoxic T cells.

Chemotherapy; chemotherapeutic agents: Any chemical agent withtherapeutic usefulness in the treatment of diseases characterized byabnormal cell growth. Such diseases include tumors, neoplasms and canceras well as diseases characterized by hyperplastic growth such aspsoriasis. In one embodiment, a chemotherapeutic agent is an agent ofuse in treating neoplasms such as solid tumors. In one embodiment, achemotherapeutic agent is a radioactive molecule. One of skill in theart can readily identify a chemotherapeutic agent of use (e.g. seeSlapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison'sPrinciples of Internal Medicine, 14th edition; Perry et al.,Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^(nd) ed., © 2000Churchill Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology PocketGuide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; FischerD S, Knobf M F, Durivage H J (eds): The Cancer Chemotherapy Handbook,4th ed. St. Louis, Mosby-Year Book, 1993). The immunogenic Brachyurypolypeptides disclosed herein can be used in conjunction with additionalchemotherapeutic agents.

Consists Essentially Of/Consists Of: With regard to a polypeptide, apolypeptide that consists essentially of a specified amino acid sequenceif it does not include any additional amino acid residues. However, thepolypeptide can include additional non-peptide components, such aslabels (for example, fluorescent, radioactive, or solid particlelabels), sugars or lipids. With regard to a polypeptide, a polypeptidethat consists of a specified amino acid sequence does not include anyadditional amino acid residues, nor does it include additionalnon-peptide components, such as lipids, sugars or labels.

Costimulatory molecule: Although engagement of the TCR with peptide-MHCdelivers one signal to the T cell, this signal alone can be insufficientto activate the T cell. Costimulatory molecules are molecules that, whenbound to their ligand, deliver a second signal required for the T cellto become activated. The most well-known costimulatory molecule on the Tcell is CD28, which binds to either B7-1 (also called CD80) or B7-2(also known as CD86). An additional costimulatory molecule is B7-3.Accessory molecules that also provide a second signal for the activationof T cells include intracellular adhesion molecule (ICAM-1 and ICAM-2),leukocyte function associated antigen (LFA-1, LFA-2 and LFA-3).Integrins and tumor necrosis factor (TNF) superfamily members can alsoserve as co-stimulatory molecules.

Degenerate variant: A polynucleotide encoding an epitope of Brachyurythat includes a sequence that is degenerate as a result of the geneticcode. There are 20 natural amino acids, most of which are specified bymore than one codon. Therefore, all degenerate nucleotide sequences areincluded in this disclosure as long as the amino acid sequence of theBrachyury polypeptide encoded by the nucleotide sequence is unchanged.

Dendritic cell (DC): Dendritic cells are the principle antigenpresenting cells (APCs) involved in primary immune responses. Dendriticcells include plasmacytoid dendritic cells and myeloid dendritic cells.Their major function is to obtain antigen in tissues, migrate tolymphoid organs and present the antigen in order to activate T cells.Immature dendritic cells originate in the bone marrow and reside in theperiphery as immature cells.

Diagnostic: Identifying the presence or nature of a pathologiccondition, such as, but not limited to, a cancer, such as smallintestine, stomach, kidney, bladder, uterus, ovary, testes, lung, colonor prostate cancer. Diagnostic methods differ in their sensitivity andspecificity. The “sensitivity” of a diagnostic assay is the percentageof diseased individuals who test positive (percent of true positives).The “specificity” of a diagnostic assay is 1 minus the false positiverate, where the false positive rate is defined as the proportion ofthose without the disease who test positive. While a particulardiagnostic method may not provide a definitive diagnosis of a condition,it suffices if the method provides a positive indication that aids indiagnosis. “Prognostic” means predicting the probability of development(for example, severity) of a pathologic condition, such as prostatecancer, or metastasis.

Epithelial-to-Mesenchymal Transition: The epithelium is the covering ofinternal and external surfaces of the body, including the lining ofvessels and other small cavities that consists of cells joined bybiological cementing substances. Generally, fully differentiatedepithelial cells express proteins characteristic of a differentiatedphenotype, such as insulin, and have a limited capacity to proliferate.The mesenchyme is the meshwork of loosely organized embryonic connectivetissue in the mesoderm from which are formed the connective tissues ofthe body, along with the blood vessels and lymphatic vessels. Vimentinis one marker of mesenchymal cells. Mesenchymal cells generally have agreater capacity to proliferate in vitro than epithelial cells and arenot fully differentiated. An “epithelial-to-mesenchymal” transition is abiological process wherein a cell, or a population of cells, from anepithelial phenotype convert to a less differentiated mesenchymalphenotype. A “mesenchymal-to-epithelial” transition is a biologicalprocess wherein a cell, or a population of cells, convert from a lessdifferentiated mesenchymal phenotype to a more differentiated epithelialphenotype.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic (that elicit aspecific immune response). An antibody specifically binds a particularantigenic epitope on a polypeptide. Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5, about 9, or 8 to 10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., “Epitope Mapping Protocols” in Methods inMolecular Biology, Vol. 66, Glenn E. Morris, Ed (1996). In oneembodiment, an epitope binds an MHC molecule, such an HLA molecule or aDR molecule. These molecules bind polypeptides having the correct anchoramino acids separated by about eight to about ten amino acids, such asnine amino acids.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus, expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see e.g., Bitter etal., Methods in Enzymology 153:516-544, 1987). For example, when cloningin bacterial systems, inducible promoters such as pL of bacteriophagelambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can beused. In one embodiment, when cloning in mammalian cell systems,promoters derived from the genome of mammalian cells (such as themetallothionein promoter) or from mammalian viruses (such as theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques can also be used to provide fortranscription of the nucleic acid sequences.

Heterologous: Originating from separate genetic sources or species. Apolypeptide that is heterologous to Brachyury originates from a nucleicacid that does not encode Brachyury. In one specific, non-limitingexample, a polypeptide comprising nine consecutive amino acids fromBrachyury, or at most 12 consecutive amino acids from Brachyury, and aheterologous amino acid sequence includes a (3-galactosidase, a maltosebinding protein, and albumin, hepatitis B surface antigen, or animmunoglobulin amino acid sequence. Generally, an antibody thatspecifically binds to a protein of interest will not specifically bindto a heterologous protein.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The cell can bemammalian, such as a human cell. The term also includes any progeny ofthe subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In one embodiment, an immune response is a T cell response,such as a CD4+ response or a CD8+ response. In another embodiment, theresponse is a B cell response, and results in the production of specificantibodies.

Immunogenic peptide: A peptide which comprises an allele-specific motifor other sequence such that the peptide will bind an MHC molecule andinduce a cytotoxic T lymphocyte (“CTL”) response, or a B cell response(e.g. antibody production) against the antigen from which theimmunogenic peptide is derived.

In one embodiment, immunogenic peptides are identified using sequencemotifs or other methods, such as neural net or polynomialdeterminations, known in the art. Typically, algorithms are used todetermine the “binding threshold” of peptides to select those withscores that give them a high probability of binding at a certainaffinity and will be immunogenic. The algorithms are based either on theeffects on MHC binding of a particular amino acid at a particularposition, the effects on antibody binding of a particular amino acid ata particular position, or the effects on binding of a particularsubstitution in a motif-containing peptide. Within the context of animmunogenic peptide, a “conserved residue” is one which appears in asignificantly higher frequency than would be expected by randomdistribution at a particular position in a peptide. In one embodiment, aconserved residue is one where the MHC structure may provide a contactpoint with the immunogenic peptide.

Immunogenic peptides can also be identified by measuring their bindingto a specific MHC protein (e.g. HLA-A02.01) and by their ability tostimulate CD4 and/or CD8 when presented in the context of the MHCprotein. The characteristics of immunogenic polypeptides, are disclosed,for example, in PCT Publication No. WO 00/12706, which is incorporatedherein by reference.

In one example, an immunogenic “Brachyury peptide” is a series ofcontiguous amino acid residues from the Brachyury protein generallybetween 7 and 20 amino acids in length, such as about 8 to 11 residuesin length. Specific immunogenic Brachyury polypeptides are disclosedherein that are 9 or 10 amino acid residues in length, or at most 12amino acids in length. Generally, immunogenic Brachyury polypeptide canbe used to induce an immune response in a subject, such as a B cellresponse or a T cell response. In one example, an immunogenic Brachyurypolypeptide, when bound to a Major Histocompatibility Complex Class Imolecule, activates cytotoxic T lymphocytes (CTLs) against cellsexpressing wild-type Brachyury protein. Induction of CTLs usingsynthetic peptides and CTL cytotoxicity assays known in the art, seeU.S. Pat. No. 5,662,907, which is incorporated herein by reference. Inone example, an immunogenic peptide includes an allele-specific motif orother sequence such that the peptide will bind an MHC molecule andinduce a cytotoxic T lymphocyte (“CTL”) response against the antigenfrom which the immunogenic peptide is derived.

Immunogenic composition: A composition comprising an immunogenicBrachyury polypeptide or a nucleic acid encoding the immunogenicBrachyury polypeptide that induces a measurable CTL response againstcells expressing Brachyury polypeptide, or induces a measurable B cellresponse (such as production of antibodies that specifically bindBrachyury) against a Brachyury polypeptide. For in vitro use, theimmunogenic composition can consist of the isolated nucleic acid, vectorincluding the nucleic acid/or immunogenic peptide. For in vivo use, theimmunogenic composition will typically comprise the nucleic acid, vectorincluding the nucleic acid, and or immunogenic polypeptide, inpharmaceutically acceptable carriers, and/or other agents. Animmunogenic composition can optionally include an adjuvant, acostimulatory molecule, or a nucleic acid encoding a costimulatorymolecule. A Brachyury polypeptide, or nucleic acid encoding thepolypeptide, can be readily tested for its ability to induce a CTL byart-recognized assays.

Inhibiting or treating a disease: Inhibiting a disease, such as tumorgrowth, refers to inhibiting the full development of a disease. Inseveral examples, inhibiting a disease refers to lessening symptoms of atumor, such as preventing the development of paraneoplastic syndrome ina person who is known to have a cancer, or lessening a sign or symptomof the tumor or reducing tumor volume. “Treatment” refers to atherapeutic intervention that ameliorates a sign or symptom of a diseaseor pathological condition related to the disease, such as the tumor.

Isolated: An “isolated” biological component (such as a nucleic acid orprotein or organelle) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs, i.e., other chromosomal andextra-chromosomal DNA and RNA, proteins and organelles. Nucleic acidsand proteins that have been “isolated” include nucleic acids andproteins purified by standard purification methods. The term alsoembraces nucleic acids and proteins prepared by recombinant expressionin a host cell as well as chemically synthesized nucleic acids.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule to facilitate detection of thatmolecule. Specific, non-limiting examples of labels include fluorescenttags, enzymatic linkages, and radioactive isotopes.

Linker sequence: A linker sequence is an amino acid sequence thatcovalently links two polypeptide domains. Linker sequences can beincluded in the between the Brachyury epitopes disclosed herein toprovide rotational freedom to the linked polypeptide domains and therebyto promote proper domain folding and presentation to the MHC. By way ofexample, in a recombinant polypeptide comprising two Brachyury domains,linker sequences can be provided between them, such as a polypeptidecomprising Brachyury polypeptide-linker-Brachyury polypeptide. Linkersequences, which are generally between 2 and 25 amino acids in length,are well known in the art and include, but are not limited to, theglycine(4)-serine spacer (GGGGS×3) described by Chaudhary et al., Nature339:394-397, 1989.

Lymphocytes: A type of white blood cell that is involved in the immunedefenses of the body. There are two main types of lymphocytes: B cellsand T cells.

Major Histocompatibility Complex (MHC): A generic designation meant toencompass the histocompatability antigen systems described in differentspecies, including the human leukocyte antigens (“HLA”).

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Neoplasm: An abnormal cellular proliferation, which includes benign andmalignant tumors, as well as other proliferative disorders.

Oligonucleotide: A linear polynucleotide sequence of up to about 100nucleotide bases in length.

Open reading frame (ORF): A series of nucleotide triplets (codons)coding for amino acids without any internal termination codons. Thesesequences are usually translatable into a peptide.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence, such as a sequence that encodes a Brachyurypolypeptide. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Peptide Modifications: Brachyury epitopes include synthetic embodimentsof peptides described herein. In addition, analogs (non-peptide organicmolecules), derivatives (chemically functionalized peptide moleculesobtained starting with the disclosed peptide sequences) and variants(homologs) of these proteins can be utilized in the methods describedherein. Each polypeptide of this disclosure is comprised of a sequenceof amino acids, which may be either L- and/or D-amino acids, naturallyoccurring and otherwise.

Peptides can be modified by a variety of chemical techniques to producederivatives having essentially the same activity as the unmodifiedpeptides, and optionally having other desirable properties. For example,carboxylic acid groups of the protein, whether carboxyl-terminal or sidechain, can be provided in the form of a salt of apharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ ester,or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ are eachindependently H or C₁-C₁₆ alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring. Amino groups of the peptide,whether amino-terminal or side chain, can be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or can be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the peptide side chains may be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chains may be substituted with one ormore halogen atoms, such as fluorine, chlorine, bromine or iodine, orwith C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acids and esters thereof,or amides of such carboxylic acids. Methylene groups of the peptide sidechains can be extended to homologous C₂-C₄ alkylenes. Thiols can beprotected with any one of a number of well-recognized protecting groups,such as acetamide groups. Those skilled in the art will also recognizemethods for introducing cyclic structures into the peptides of thisinvention to select and provide conformational constraints to thestructure that result in enhanced stability.

Peptidomimetic and organomimetic embodiments are envisioned, whereby thethree-dimensional arrangement of the chemical constituents of suchpeptido- and organomimetics mimic the three-dimensional arrangement ofthe peptide backbone and component amino acid side chains, resulting insuch peptido- and organomimetics of an immunogenic Brachyury polypeptidehaving measurable or enhanced ability to generate an immune response.For computer modeling applications, a pharmacophore is an idealizedthree-dimensional definition of the structural requirements forbiological activity. Peptido- and organomimetics can be designed to fiteach pharmacophore with current computer modeling software (usingcomputer assisted drug design or CADD). See Walters, “Computer-AssistedModeling of Drugs,” in Klegennan & Groves, eds., 1993, PharmaceuticalBiotechnology, Interpharm Press: Buffalo Grove, Ill., pp. 165-174 andPrinciples of Pharmacology, Munson (ed.) 1995, Ch. 102, for descriptionsof techniques used in CADD. Also included are mimetics prepared usingsuch techniques.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975),describes compositions and formulations suitable for pharmaceuticaldelivery of the fusion proteins herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (such as powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

A “therapeutically effective amount” is a quantity of a composition or acell to achieve a desired effect in a subject being treated. Forinstance, this can be the amount necessary to induce an immune response,inhibit tumor growth, reduce tumor volume or to measurably alter outwardsymptoms of the tumor. When administered to a subject, a dosage willgenerally be used that will achieve target tissue concentrations (forexample, in lymphocytes) that has been shown to achieve an in vitroeffect.

Polynucleotide: The term polynucleotide or nucleic acid sequence refersto a polymeric form of nucleotide at least 10 bases in length. Arecombinant polynucleotide includes a polynucleotide that is notimmediately contiguous with both of the coding sequences with which itis immediately contiguous (one on the 5′ end and one on the 3′ end) inthe naturally occurring genome of the organism from which it is derived.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (e.g., a cDNA) independent of othersequences. The nucleotides can be ribonucleotides, deoxyribonucleotides,or modified forms of either nucleotide. The term includes single- anddouble-stranded forms of DNA.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). In one embodiment, the polypeptide is a Brachyurypolypeptide. A polypeptide can be between 3 and 30 amino acids inlength. In one embodiment, a polypeptide is from about 7 to about 25amino acids in length. In yet another embodiment, a polypeptide is fromabout 8 to about 12 amino acids in length. In yet another embodiment, apeptide is about 9 amino acids in length. With regard to polypeptides,the word “about” indicates integer amounts. Thus, in one example, apolypeptide “about” 9 amino acids in length is from 8 to 10 amino acidsin length.

Probes and primers: A probe comprises an isolated nucleic acid attachedto a detectable label or reporter molecule. Primers are short nucleicacids, preferably DNA oligonucleotides, of about 15 nucleotides or morein length. Primers may be annealed to a complementary target DNA strandby nucleic acid hybridization to form a hybrid between the primer andthe target DNA strand, and then extended along the target DNA strand bya DNA polymerase enzyme. Primer pairs can be used for amplification of anucleic acid sequence, for example by polymerase chain reaction (PCR) orother nucleic-acid amplification methods known in the art. One of skillin the art will appreciate that the specificity of a particular probe orprimer increases with its length. Thus, for example, a primer comprising20 consecutive nucleotides will anneal to a target with a higherspecificity than a corresponding primer of only 15 nucleotides. Thus, inorder to obtain greater specificity, probes and primers can be selectedthat comprise about 20, 25, 30, 35, 40, 50 or more consecutivenucleotides.

Purified: The epitopes of Brachyury disclosed herein can be purified(and/or synthesized) by any of the means known in the art (see, e.g.,Guide to Protein Purification, ed. Deutscher, Meth. Enzymol. 185,Academic Press, San Diego, 1990; and Scopes, Protein Purification:Principles and Practice, Springer Verlag, New York, 1982). Substantialpurification denotes purification from other proteins or cellularcomponents. A substantially purified protein is at least about 60%, 70%,80%, 90%, 95%, 98% or 99% pure. Thus, in one specific, non-limitingexample, a substantially purified protein is 90% free of other proteinsor cellular components.

Thus, the term purified does not require absolute purity; rather, it isintended as a relative term. For example, a purified nucleic acid is onein which the nucleic acid is more enriched than the nucleic acid in itsnatural environment within a cell. In additional embodiments, a nucleicacid or cell preparation is purified such that the nucleic acid or cellrepresents at least about 60% (such as, but not limited to, 70%, 80%,90%, 95%, 98% or 99%) of the total nucleic acid or cell content of thepreparation, respectively.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Selectively hybridize: Hybridization under moderately or highlystringent conditions that excludes non-related nucleotide sequences.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (for example, GC v. ATcontent), and nucleic acid type (for example, RNA versus DNA) of thehybridizing regions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

A specific example of progressively higher stringency conditions is asfollows: 2×SC/0.1% SDS at about room temperature (hybridizationconditions); 0.2×SC/0.1% SDS at about room temperature (low stringencyconditions); 0.2×SC/0.1% SDS at about 42° C. (moderate stringencyconditions); and 0.1×SC at about 68° C. (high stringency conditions).One of skill in the art can readily determine variations on theseconditions (e.g., Molecular Cloning: A Laboratory Manual, 2nd ed., vol.1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989). Washing can be carried out using only one ofthese conditions, e.g., high stringency conditions, or each of theconditions can be used, e.g., for 10-15 minutes each, in the orderlisted above, repeating any or all of the steps listed. However, asmentioned above, optimal conditions will vary, depending on theparticular hybridization reaction involved, and can be determinedempirically.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of a Brachyury polypeptide will possess arelatively high degree of sequence identity when aligned using standardmethods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Higgins and Sharp, Gene 73:237, 1988; Higginsand Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a Brachyury polypeptide are typicallycharacterized by possession of at least 75%, for example at least 80%,sequence identity counted over the full length alignment with the aminoacid sequence of Brachyury using the NCBI Blast 2.0, gapped blastp setto default parameters. For comparisons of amino acid sequences ofgreater than about 30 amino acids, the Blast 2 sequences function isemployed using the default BLOSUM62 matrix set to default parameters,(gap existence cost of 11, and a per residue gap cost of 1). Whenaligning short peptides (fewer than around 30 amino acids), thealignment should be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties). Proteins with even greater similarity to thereference sequences will show increasing percentage identities whenassessed by this method, such as at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% sequence identity. Whenless than the entire sequence is being compared for sequence identity,homologs and variants will typically possess at least 80% sequenceidentity over short windows of 10-20 amino acids, and can possesssequence identities of at least 85% or at least 90% or 95% depending ontheir similarity to the reference sequence. Methods for determiningsequence identity over such short windows are available at the NCBIwebsite on the internet. One of skill in the art will appreciate thatthese sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs could be obtainedthat fall outside of the ranges provided.

Small interfering RNAs: Synthetic or naturally-produced small doublestranded RNAs (dsRNAs) that can induce gene-specific inhibition ofexpression in invertebrate and vertebrate species are provided. TheseRNAs are suitable for interference or inhibition of expression of atarget gene and comprise double stranded RNAs of about 15 to about 40nucleotides containing a 3′ and/or 5′ overhang on each strand having alength of 0- to about 5-nucleotides, wherein the sequence of the doublestranded RNAs is essentially identical to a portion of a coding regionof the target gene for which interference or inhibition of expression isdesired. The double stranded RNAs can be formed from complementaryssRNAs or from a single stranded RNA that forms a hairpin or fromexpression from a DNA vector.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a Brachyury specific binding agent is an agent thatbinds substantially to a Brachyury polypeptide. In one embodiment, thespecific binding agent is a monoclonal or polyclonal antibody thatspecifically binds Brachyury.

T Cell: A white blood cell critical to the immune response. T cellsinclude, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ Tlymphocyte is an immune cell that carries a marker on its surface knownas “cluster of differentiation 4” (CD4). These cells, also known ashelper T cells, help orchestrate the immune response, including antibodyresponses as well as killer T cell responses. CD8⁺ T cells carry the“cluster of differentiation 8” (CD8) marker. In one embodiment, a CD8 Tcell is a cytotoxic T lymphocyte. In another embodiment, a CD8 cell is asuppressor T cell.

Therapeutically active polypeptide: An agent, such as an epitope ofBrachyury that causes induction of an immune response, as measured byclinical response (for example increase in a population of immune cells,increased cytolytic activity against cells that express Brachyury, ormeasurable reduction of tumor burden). Therapeutically active moleculescan also be made from nucleic acids. Examples of a nucleic acid basedtherapeutically active molecule is a nucleic acid sequence that encodesa Brachyury epitope, wherein the nucleic acid sequence is operablylinked to a control element such as a promoter. Another example of atherapeutically active molecule is an antisense molecule or a siRNA forBrachyury.

In one embodiment, a therapeutically effective amount of a composition,such as a Brachyury polypeptide, is an amount used to generate an immuneresponse, or to treat cancer in a subject. In several examples,“treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a cancer, or a reduction in tumor burden.

Transduced: A transduced cell is a cell into which has been introduced anucleic acid molecule by molecular biology techniques. As used herein,the term transduction encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergene and other genetic elements known in the art. Vectors includeplasmid vectors, including plasmids for expression in gram negative andgram positive bacterial cell. Exemplary vectors include those forexpression in E. coli and Salmonella. Vectors also include viralvectors, such as, but are not limited to, retrovirus, orthopox, avipox,fowlpox, capripox, suipox, adenoviral, herpes virus, alpha virus,baculovirus, Sindbis virus, vaccinia virus and poliovirus vectors.Vectors also include vectors for expression in yeast cells.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

Immunogenic Brachyury Peptides

Brachyury (also known as “T-protein”) is a polypeptide which istranscribed in the mesoderm. In one embodiment, the polypeptide has asequence set forth as:

(SEQ ID NO: 1) MSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTERELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERSDHKEMMEEPGDSQQPGYSQWGWLLPGTSTLCPPANPHPQFGGALSLPSTHSCDRYPTLRSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLGMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTPGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGSPLYEGAAAATDIVDSQYDAAAQGRLIASWTPVSPPSM (see also GENBANK® Accession No NP_(—)003172 and GENBANK® Accession No.NM_(—)003181, as available on Feb. 23, 2007, incorporated herein byreference).

In other embodiments, Brachyury has an amino acid sequence at least 90%identical to SEQ ID NO: 1, for example a polypeptide that has at leastabout 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity toSEQ ID NO: 1.

Using the genetic code, one of skill in the art can readily produce anucleic acid sequence encoding Brachyury. In one example, Brachyury isencoded by a nucleic acid having a sequence set forth as:

(SEQ ID NO: 1)tttgcttttg cttatttccg tccatttccc tctctgcgcg cggaccttcc ttttccagatggtgagagcc gcggggacac ccgacgccgg ggcaggctga tccacgatcc tgggtgtgcgtaacgccgcc tggggctccg tgggcgaggg acgtgtgggg acaggtgcac cggaaactgccagactggag agttgaggca tcggaggcgc gagaacagca ctactactgc ggcgagacgagcgcggcgca tcccaaagcc cggccaaatg cgctcgtccc tgggagggga gggaggcgcgcctggagcgg ggacagtctt ggtccgcgcc ctcctcccgg gtctgtgccg ggacccgggacccgggagcc gtcgcaggtc tcggtccaag gggccccttt tctcggaagg gcggcggccaagagcaggga aggtggatct caggtagcga gtctgggctt cggggacggc ggggaggggagccggacggg aggatgagct cccctggcac cgagagcgcg ggaaagagcc tgcagtaccgagtggaccac ctgctgagcg ccgtggagaa tgagctgcag gcgggcagcg agaagggcgaccccacagag cgcgaactgc gcgtgggcct ggaggagagc gagctgtggc tgcgcttcaaggagctcacc aatgagatga tcgtgaccaa gaacggcagg aggatgtttc cggtgctgaaggtgaacgtg tctggcctgg accccaacgc catgtactcc ttcctgctgg acttcgtggcggcggacaac caccgctgga agtacgtgaa cggggaatgg gtgccggggg gcaagccggagatgaaggct cccgtctcct tcagcaaagt caagctcacc aacaagctca acggagggggccagatcatg ctgaactcct tgcataagta tgagcctcga atccacatag tgagagttgggggtccacag cgcatgatca ccagccactg cttccctgag acccagttca tagcggtgactgcttatcag aacgaggaga tcacagctct taaaattaag tacaatccat ttgcaaaagctttccttgat gcaaaggaaa gaagtgatca caaagagatg atggaggaac ccggagacagccagcaacct gggtactccc aatgggggtg gcttcttcct ggaaccagca ccctgtgtccacctgcaaat cctcatcctc agtttggagg tgccctctcc ctcccctcca cgcacagctgtgacaggtac ccaaccctga ggagccaccg gtcctcaccc taccccagcc cctatgctcatcggaacaat tctccaacct attctgacaa ctcacctgca tgtttatcca tgctgcaatcccatgacaat tggtccagcc ttggaatgcc tgcccatccc agcatgctcc ccgtgagccacaatgccagc ccacctacca gctccagtca gtaccccagc ctgtggtctg tgagcaacggcgccgtcacc ccgggctccc aggcagcagc cgtgtccaac gggctggggg cccagttcttccggggctcc cccgcgcact acacacccct cacccatccg gtctcggcgc cctcttcctcgggatcccca ctgtacgaag gggcggccgc ggccacagac atcgtggaca gccagtacgacgccgcagcc caaggccgcc tcatagcctc atggacacct gtgtcgccac cttccatgtgaagcagcaag gcccaggtcc cgaaagatgc agtgactttt tgtcgtggca gccagtggtgactggattga cctactaggt acccagtggc agtctcaggt taagaaggaa atgcagcctcagtaacttcc ttttcaaagc agtggaggag cacacggcac ctttccccag agccccagcatcccttgctc acacctgcag tagcggtgct gtcccaggtg gcttacagat gaacccaactgtggagatga tgcagttggc ccaacctcac tgacggtgaa aaaatgtttg ccagggtccagaaacttttt ttggtttatt tctcatacag tgtattggca actttggcac accagaatttgtaaactcca ccagtcctac tttagtgaga taaaaagcac actcttaatc ttcttccttgttgctttcaa gtagttagag ttgagctgtt aaggacagaa taaaatcata gttgaggacagcaggtttta gttgaattga aaatttgact gctctgcccc ctagaatgtg tgtattttaagcatatgtag ctaatctctt gtgttgttaa actataactg tttcatattt ttcttttgacaaagtagcca aagacaatca gcagaaagca ttttctgcaa aataaacgca atatgcaaaatgtgattcgt ccagttatta gtgaagcccc tccttttgtg agtatttact gtttattg

Immunogenic fragments of Brachyury (and Brachyury itself), can bechemically synthesized by standard methods. If desired, polypeptides canalso be chemically synthesized by emerging technologies. One suchprocess is described in W. Lu et al., Federation of European BiochemicalSocieties Letters. 429:31-35, 1998. Polypeptides can also be producedusing molecular genetic techniques, such as by inserting a nucleic acidencoding Brachyury or an epitope thereof into an expression vector,introducing the expression vector into a host cell, and isolating thepolypeptide (see below).

Brachyury polypeptides are disclosed herein that can be used to inducean immune response (are immunogenic). These peptides comprise at mosttwelve amino acids, such as eleven, ten amino acids, or nine consecutiveamino acids of a Brachyury polypeptide.

An isolated polypeptide is disclosed that includes at most twelveconsecutive amino acids from Brachyury, wherein the isolated polypeptidecomprises the amino acid sequence set forth as WLLPGTSTX₁ (SEQ ID NO:3), wherein X₁ is a leucine (L) or a valine (V). In some embodiments,amino acid 1 (X₁) is a leucine. In additional embodiments, amino acid 1(X₁) is a valine. In one example the polypeptide consists essentially ofthe amino acid sequence set forth as SEQ ID NO: 3. Thus, in one example,the polypeptide consists essentially of SEQ ID NO: 3, wherein amino acidX₁ is a valine (V), and in another example the polypeptide consistsessentially of SEQ ID NO: 3, wherein amino acid X₁ is a leucine. Inadditional examples, the polypeptide is eleven amino acids in length orten amino acids in length. In further examples, the isolated polypeptideconsists of the amino acid sequence set forth as SEQ ID NO: 3.

In additional embodiments the isolated Brachyury polypeptide is nine totwelve amino acids in length and comprises the amino acid sequence setforth as SQYPSLWSV (SEQ ID NO: 14), WLLPGTSTL (SEQ ID NO: 15), RLIASWTPV(SEQ ID NO: 16), or AMYSFLLDFV (SEQ ID NO: 17). In several examples, theisolated Brachyury polypeptide is nine or ten amino acids in length, andcomprises one of the amino acid sequences set forth as SEQ ID NOs:14-17. In additional examples, the isolated Brachyury polypeptideconsists of the amino acid sequence set forth as one of SEQ ID NO:14-17.

In additional embodiments, the Brachyury polypeptide is nine to twelveamino acids in length, and comprises the amino acid sequence:SX₂YX₃SLX₄SX₅ (SEQ ID NO: 18), wherein X₂ and X₅ are either a valine ora leucine, wherein X₃ is proline (P), serine (S), threonine (T), leucine(L), or valine (V) and wherein X₄ is tryptophan (W), valine (V), leucine(L), isoleucine (I), serine (S) or threorine (T). In further examples,the Brachyury polypeptide is nine, ten or eleven amino acids in length,and comprises the amino acid sequence set forth as SEQ ID NO: 18,wherein X₅ is one of valine or a leucine. In additional examples, theBrachyury polypeptide consists of the amino acid sequence set forth asSEQ ID NO: 18 wherein X₅ is a valine or a leucine. The followingexemplary Brachyury polypeptides are encompassed by the presentdisclosure:

T-p1a: SLYPSLWSV (SEQ ID NO: 18, wherein X₂ is L, X₃ is P, X₄ is Wand X₅ is V) T-p1b: SLYPSLWSL(SEQ ID NO: 18, wherein X₂ is L, X₃ is P, X₄ is W and X₅ is L)T-p1c: SX₂YSSLWSV (SEQ ID NO: 18, wherein X₂ is Q or L, X₃ is Y, X₄is W and X₅ is V) T-p1d: SX₂YTSLWSV(SEQ ID NO: 18, wherein X₂ is Q or L, X₃ is T, X₄ is W, and X₅ is V)T-p1e: SX₂YLSLWSV (SEQ ID NO: 18, wherein X₂ is Q or L, X₃ is L, X₄is W and X₅ is V) T-p1f: SX₂YVSLWSV(SEQ ID NO: 18, wherein X₂ is Q or L, X₃ is V, X₄ is W and X₅ is V)T-p1g: SX₂YPSLVSV (SEQ ID NO: 18, wherein X₂ is Q or L, X₃ is P, X₄is V and X₅ is V) T-p1h: SX₂YPSLLSV(SEQ ID NO: 18, wherein X₂ is Q or L, X₃ is P, X₄ is L and X₅ is V)T-p1i: SX₂YPSLISV (SEQ ID NO: 18, wherein X₂ is Q or L, X₃ is P, X₄is I and X₅ is V) T-p1j: SX₂YPSLSSV(SEQ ID NO: 18, wherein X₂ is Q or L, X₃ is P, X₄ is S and X₅ is V)T-p1k: SX₂YPSLTSV (SEQ ID NO: 18, wherein X₂ is Q or L, X₃ is P, X₄is T and X₅ is V)

In further embodiments, the Brachyury polypeptide is nine to twelveamino acids in length, and comprises the amino acid sequence:WLLX₆GTSTX₇ (SEQ ID NO: 19), wherein X₆ is serine (S), threonine (T),isoleucine (I), valine (V) and wherein X₇ is leucine (L) or valine. Infurther examples, the Brachyury polypeptide is nine or ten amino acidsin length, and comprises the amino acid sequence set forth as SEQ ID NO:19, wherein X₇ is one of valine or a leucine. In additional examples,the Brachyury polypeptide consists of the amino acid sequence set forthas SEQ ID NO: 19 wherein X₅₋₇ is a valine or a leucine. The followingexemplary polypeptides are encompassed by this disclosure:

Tp2b: WLLSGTSTX₇ (SEQ ID NO: 19, wherein X₆ is S, and X₇ is L or V)Tp2c: WLLTGTSTX₇ (SEQ ID NO: 19, wherein X₆ is T, and X₇ is L or V)Tp2d: WLLIGTSTX₇ (SEQ ID NO: 19, wherein X₆ is I, and X₇ is L or V)Tp2e: WLLVGTSTX₇ (SEQ ID NO: 19, wherein X₆ is V, and X₇ is L or V)

In additional embodiments, the Brachyury polypeptide is nine to twelveamino acids in length, and comprises the amino acid sequence X₈LIASTTPV(SEQ ID NO: 20), wherein X₈ is tyrosine (Y) or tryptophan (W). TheBrachyury polypeptide can be nine or ten amino acids in length, andcomprises the amino acid sequence set forth as X₈LIASTTPV (SEQ ID NO:20, wherein X₈ is one of tyrosine (Y) or tryptophan (W). In additionalexamples, the Brachyury polypeptide consists of the amino acid sequenceset forth as SEQ ID NO: 20 wherein X₈ is a tryptophan or a tyrosine.Thus, the following polypeptides are encompassed by the presentdisclosure:

T-p3a: YLIASWTPV (SEQ ID NO: 20, wherein X₈ is Y) T-p3b: WLIASWTPV(SEQ ID NO: 20, wherein X₈ is W)

In another set of embodiments, the isolated Brachyury polypeptide isnine to twelve amino acids in length, and comprises the amino acidsequence: X₉LIASX₁₀TPV (SEQ ID NO: 21), wherein X₉ is an arginine (R),tyrosine (Y) or tryptophan (W) and X₁₀ is a valine (V), lysine (L),isoleucine (I), serine (S) or threonine (T). In some examples, theisolated Brachyury polypeptide is nine or ten amino acids in length, andcomprises the amino acid sequence set forth as X₉LIASTTPV (SEQ ID NO:21, X₉ is an arginine, tyrosine or tryptophan and X₁₀ is a valine,lysine, isoleucine, serine or threonine). In additional examples, theBrachyury polypeptide consists of the amino acid sequence set forth asSEQ ID NO: 21 wherein X₉ is an arginine, tyrosine or tryptophan and X₁₀is a valine, serine, isoleucine, or threonine. Thus, the followingpolypeptides are encompassed by the present disclosure:

Tp3c: X₉LIASVTPV (SEQ ID NO: 21, wherein X₉ = R, Y, or W And X₁₀ is V)Tp3d: X₉LIASLTPV (SEQ ID NO: 21, wherein X₉ = R, Y, or W And X₁₀ is V)Tp3e: X₉LIASITPV (SEQ ID NO: 21, wherein X₉ = R, Y, or W And X₁₀ is V)Tp3f: X₉LIASSTPV (SEQ ID NO: 21, wherein X₉ = R, Y, or W And X₁₀ is V)Tp3g: X₉LIASTTPV (SEQ ID NO: 21, wherein X₉ = R, Y, or W And X₁₀ is V)

In an additional embodiment, the isolated Brachyury polypeptide is tento twelve amino acids in length, and comprises the amino acid sequenceALYSFLLDFV (SEQ ID NO: 22, T-p4a). In some examples, the isolatedBrachyury polypeptide is ten or eleven amino acids in length andcomprises ALYSFLLDFV (SEQ ID NO: 22). In an additional example, theisolated Brachyury polypeptide consists of ALYSFLLDFV (SEQ ID NO: 22).

In several embodiments, the isolated Brachyury polypeptide is include ina fusion protein. Thus, the fusion protein can include the Brachyurypolypeptide (see above) and a second heterologous moiety, such as a mycprotein, an enzyme or a carrier (such as a hepatitis carrier protein orbovine serum albumin) covalently linked to the Brachyury polypeptide. Inadditional embodiments, the protein consists of the Brachyurypolypeptide. Thus, a second heterologous moiety is non-covalently linkedto the Brachyury polypeptide. For example, the polypeptide can be nineor ten acid amino acids in length, and consists of the sequence setforth as one of SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ IDNO: 21, SEQ ID NO: 22.

These above-described brachyury polypeptides are immunogenic, and thuscan be used to induce an immune response in a subject. The Brachyurypolypeptides disclosed herein do not include all the additionalconsecutive amino acids of SEQ ID NO: 1. In one embodiment, thepolypeptide does not include amino acids 1-15 of SEQ ID NO: 1.

Without being bound by theory, it is believed that the presentation ofpeptides by MHC Class I molecules involves binding to the cleft in anMHC Class I molecule through the anchor residues of the peptide andultimate presentation on the cell surface. Depending upon the particularanchor residues, among other things, certain peptides can bind moretightly to particular HLA molecules than others. Peptides that bind wellare usually “dominant” epitopes, while those that bind less well areoften “subdominant” or “cryptic” epitopes. Dominant epitopes of eitherself proteins or foreign proteins evoke strong tolerance or immuneresponses. Subdominant or cryptic epitopes generate weak responses or noresponses at all. Without being bound by theory, tighter binding bydominant epitopes to HLA molecules results in their denser presentationon the cell surface, greater opportunity to react with immune cells andgreater likelihood of eliciting an immune response or tolerance. MHCClass I molecules present epitopes from endogenous proteins forpresentation to CTL cells. HLA A, HLA B and HLA C molecules bindpeptides of about eight to ten amino acids in length (such as nine aminoacids in length) that have particular anchoring residues. The anchoringresidues recognized by an HLA Class I molecule depend upon theparticular allelic form of the HLA molecule. A CD8+ T cell bears T cellreceptors that recognize a specific epitope when presented by aparticular HLA molecule on a cell. When a CTL precursor that has beenstimulated by an antigen presenting cell to become a cytotoxic Tlymphocyte contacts a cell that bears such an HLA-peptide complex, theCTL forms a conjugate with the cell and destroys it. In several examplespresented herein, the polypeptides that are disclosed bind and arepresented by HLA-A2.1.

In several examples, the Brachyury polypeptide can be repeated inseries, such that the polypeptide includes several copies of theimmunogenic Brachyury polypeptide. However, only one copy of theBrachyury polypeptide can be included in an immunogenic molecule. Inseveral examples, two, three, four, five copies of the Brachyurypolypeptide are included in an immunogenic molecule. The copies of theBrachyury polypeptide can be separated by peptide linkers.

In additional examples, the polypeptide can be a fusion protein and canalso include heterologous sequences to Brachyury (such as amino acidsequences of at least nine amino acids in length that are not includedin SEQ ID NO: 1). Thus, in several specific non-limiting examples, theimmunogenic peptide is a fusion polypeptide, for example the polypeptideincludes six sequential histidine residues, a β-galactosidase amino acidsequence, or an immunoglobulin amino acid sequence. The polypeptide canalso be covalently linked to a carrier. Suitable carriers include, butare not limited to, a hepatitis B small envelope protein HBsAg. Thisprotein has the capacity to self assemble into aggregates and can formviral-like particles. The preparation of HBsAg is well documented, seefor example European Patent Application Publication No. EP-A-0 226 846,European Patent Application Publication No. EP-A-0 299 108 and PCTPublication No. WO 01/117554, and the amino acid sequence disclosed, forexample, in Tiollais et al., Nature, 317: 489, 1985, and European PatentPublication No. EP-A-0 278 940, and PCT Publication No. WO 91/14703, allof which are incorporated herein by reference.

As noted above, the fusion polypeptide can optionally includerepetitions of one or more of the Brachyury polypeptides disclosedherein. In one specific, non-limiting example, the polypeptide includestwo, three, four, five, or up to ten repetitions of one of a Brachyurypolypeptide. A linker sequence can optionally be included between theBrachyury polypeptides. In all of these examples, the polypeptide doesnot include the full-length Brachyury amino acid sequence, such as theamino acid sequence set forth as SEQ ID NO: 1.

The Brachyury polypeptides disclosed herein can be chemicallysynthesized by standard methods, or can be produced recombinantly. Anexemplary process for polypeptide production is described in Lu et al.,Federation of European Biochemical Societies Letters. 429:31-35, 1998.They can also be isolated by methods including preparativechromatography and immunological separations.

A Brachyury polypeptide can be covalently linked to a carrier, which isan immunogenic macromolecule to which an antigenic molecule can bebound. When bound to a carrier, the bound polypeptide becomes moreimmunogenic. Carriers are chosen to increase the immunogenicity of thebound molecule and/or to elicit higher titers of antibodies against thecarrier which are diagnostically, analytically, and/or therapeuticallybeneficial. Covalent linking of a molecule to a carrier can conferenhanced immunogenicity and T cell dependence (see Pozsgay et al., PNAS96:5194-97, 1999; Lee et al., J. Immunol. 116:1711-18, 1976; Dintzis etal., PNAS 73:3671-75, 1976). Useful carriers include polymeric carriers,which can be natural (for example, polysaccharides, polypeptides orproteins from bacteria or viruses), semi-synthetic or syntheticmaterials containing one or more functional groups to which a reactantmoiety can be attached. Bacterial products and viral proteins (such ashepatitis B surface antigen and core antigen) can also be used ascarriers, as well as proteins from higher organisms such as keyholelimpet hemocyanin, horseshoe crab hemocyanin, edestin, mammalian serumalbumins, and mammalian immunoglobulins. Additional bacterial productsfor use as carriers include bacterial wall proteins and other products(for example, streptococcal or staphylococcal cell walls andlipopolysaccharide (LPS)).

Polynucleotides encoding the Brachyury polypeptides disclosed herein arealso provided. These polynucleotides include DNA, cDNA and RNA sequenceswhich encode the polypeptide of interest. Silent mutations in the codingsequence result from the degeneracy (i.e., redundancy) of the geneticcode, whereby more than one codon can encode the same amino acidresidue. Thus, for example, leucine can be encoded by CTT, CTC, CTA,CTG, TTA, or TTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, orAGC; asparagine can be encoded by AAT or AAC; aspartic acid can beencoded by GAT or GAC; cysteine can be encoded by TGT or TGC; alaninecan be encoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAAor CAG; tyrosine can be encoded by TAT or TAC; and isoleucine can beencoded by ATT, ATC, or ATA. Tables showing the standard genetic codecan be found in various sources (e.g., L. Stryer, 1988, Biochemistry,3.sup.rd Edition, W.H. 5 Freeman and Co., NY).

A nucleic acid encoding a Brachyury polypeptide can be cloned oramplified by in vitro methods, such as the polymerase chain reaction(PCR), the ligase chain reaction (LCR), the transcription-basedamplification system (TAS), the self-sustained sequence replicationsystem (3SR) and the Q13 replicase amplification system (QB). Forexample, a polynucleotide encoding the protein can be isolated bypolymerase chain reaction of cDNA using primers based on the DNAsequence of the molecule. A wide variety of cloning and in vitroamplification methodologies are well known to persons skilled in theart. PCR methods are described in, for example, U.S. Pat. No. 4,683,195;Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; andErlich, ed., PCR Technology, (Stockton Press, NY, 1989). Polynucleotidesalso can be isolated by screening genomic or cDNA libraries with probesselected from the sequences of the desired polynucleotide understringent hybridization conditions.

The polynucleotides encoding a Brachyury polypeptide include arecombinant DNA which is incorporated into a vector in an autonomouslyreplicating plasmid or virus or into the genomic DNA of a prokaryote oreukaryote, or which exists as a separate molecule (such as a cDNA)independent of other sequences. The nucleotides of the invention can beribonucleotides, deoxyribonucleotides, or modified focus of eithernucleotide. The term includes single and double forms of DNA.

In one embodiment, vectors are used for expression in yeast such as S.cerevisiae or Kluyveromyces lactis. Several promoters are known to be ofuse in yeast expression systems such as the constitutive promotersplasma membrane H⁺-ATPase (PMA1), glyceraldehyde-3-phosphatedehydrogenase (GPD), phosphoglycerate kinase-1 (PGK1), alcoholdehydrogenase-1 (ADM), and pleiotropic drug-resistant pump (PDR5). Inaddition, may inducible promoters are of use, such as GAL1-10 (inducedby galactose), PHO5 (induced by low extracellular inorganic phosphate),and tandem heat shock HSE elements (induced by temperature elevation to37° C.). Promoters that direct variable expression in response to atitratable inducer include the methionine-responsive MET3 and MET25promoters and copper-dependent CUP1 promoters. Any of these promotersmay be cloned into multicopy (2μ) or single copy (CEN) plasmids to givean additional level of control in expression level. The plasmids caninclude nutritional markers (such as URA3, ADE3, HIS1, and others) forselection in yeast and antibiotic resistance (AMP) for propagation inbacteria. Plasmids for expression on K. lactis are known, such aspKLAC 1. Thus, in one example, after amplification in bacteria, plasmidscan be introduced into the corresponding yeast auxotrophs by methodssimilar to bacterial transformation.

The brachyury peptides can be expressed in a variety of yeast strains.For example, seven pleiotropic drug-resistant transporters, YOR1, SNQ2,PDR5, YCF1, PDR10, PDR11, and PDR15, together with their activatingtranscription factors, PDR1 and PDR3, have been simultaneously deletedin yeast host cells, rendering the resultant strain sensitive to drugs.Yeast strains with altered lipid composition of the plasma membrane,such as the erg6 mutant defective in ergosterol biosynthesis, can alsobe utilized. Proteins that are highly sensitive to proteolysis can beexpressed in a yeast lacking the master vacuolar endopeptidase Pep4,which controls the activation of other vacuolar hydrolases. Heterologousexpression in strains carrying temperature-sensitive (ts) alleles ofgenes can be employed if the corresponding null mutant is inviable.

Viral vectors can also be prepared encoding the brachyury polypeptidesdisclosed herein. A number of viral vectors have been constructed,including polyoma, SV40 (Madzak et al., 1992, J. Gen. Virol.,73:15331536), adenovirus (Berkner, 1992, Cur. Top. Microbiol. Immunol.,158:39-6; Berliner et al., 1988, Bio Techniques, 6:616-629; Gorziglia etal., 1992, J. Virol., 66:4407-4412; Quantin et al., 1992, Proc. Nad.Acad. Sci. USA, 89:2581-2584; Rosenfeld et al., 1992, Cell, 68:143-155;Wilkinson et al., 1992, Nucl. Acids Res., 20:2233-2239;Stratford-Perricaudet et al., 1990, Hum. Gene Ther., 1:241-256),vaccinia virus (Mackett et al., 1992, Biotechnology, 24:495-499),adeno-associated virus (Muzyczka, 1992, Curr. Top. Microbiol. Immunol.,158:91-123; On et al., 1990, Gene, 89:279-282), herpes viruses includingHSV and EBV (Margolskee, 1992, Curr. Top. Microbiol. Immunol.,158:67-90; Johnson et al., 1992, J. Virol., 66:29522965; Fink et al.,1992, Hum. Gene Ther. 3:11-19; Breakfield et al., 1987, Mol. Neurobiol.,1:337-371; Fresse et al., 1990, Biochem. Pharmacol., 40:2189-2199),Sindbis viruses (H. Herweijer et al., 1995, Human Gene Therapy6:1161-1167; U.S. Pat. Nos. 5,091,309 and 5,2217,879), alphaviruses (S.Schlesinger, 1993, Trends Biotechnol. 11:18-22; I. Frolov et al., 1996,Proc. Natl. Acad. Sci. USA 93:11371-11377) and retroviruses of avian(Brandyopadhyay et al., 1984, Mol. Cell Biol., 4:749-754; Petropouploset al., 1992, J. Virol., 66:3391-3397), murine (Miller, 1992, Curr. Top.Microbiol. Immunol., 158:1-24; Miller et al., 1985, Mol. Cell Biol.,5:431-437; Sorge et al., 1984, Mol. Cell Biol., 4:1730-1737; Mann etal., 1985, J. Virol., 54:401-407), and human origin (Page et al., 1990,J. Virol., 64:5370-5276; Buchschalcher et al., 1992, J. Virol.,66:2731-2739). Baculovirus (Autographa californica multinuclearpolyhedrosis virus; AcMNPV) vectors are also known in the art, and maybe obtained from commercial sources (such as PharMingen, San Diego,Calif.; Protein Sciences Corp., Meriden, Conn.; Stratagene, La Jolla,Calif.).

Thus, in one embodiment, the polynucleotide encoding a Brachyurypolypeptide is included in a viral vector. Suitable vectors includeretrovirus vectors, orthopox vectors, avipox vectors, fowlpox vectors,capripox vectors, suipox vectors, adenoviral vectors, herpes virusvectors, alpha virus vectors, baculovirus vectors, Sindbis virusvectors, vaccinia virus vectors and poliovirus vectors. Specificexemplary vectors are poxvirus vectors such as vaccinia virus, fowlpoxvirus and a highly attenuated vaccinia virus (MVA), adenovirus,baculovirus and the like.

Pox viruses useful in practicing the present invention include orthopox,suipox, avipox, and capripox virus. Orthopox include vaccinia,ectromelia, and raccoon pox. One example of an orthopox of use isvaccinia. Avipox includes fowlpox, canary pox and pigeon pox. Capripoxinclude goatpox and sheeppox. In one example, the suipox is swinepox.Examples of pox viral vectors for expression as described for example,in U.S. Pat. No. 6,165,460, which is incorporated herein by reference.Other viral vectors that can be used include other DNA viruses such asherpes virus and adenoviruses, and RNA viruses such as retroviruses andpolio.

In some cases, vaccinia viral vectors may elicit a strong antibodyresponse. Thus, while numerous boosts with vaccinia vectors arepossible, its repeated use may not be useful in certain instances.However, this sensitivity problem can be minimized by using pox fromdifferent genera for boosts. In one example, when the first or initialpox virus vector is vaccinia, the second and subsequent pox virusvectors are selected from the pox viruses from a different genus such assuipox, avipox, capripox or an orthopox immunogenically distinct fromvaccinia.

The vaccinia virus genome is known in the art. It is composed of a HINDF13L region, TK region, and an HA region. Recombinant vaccinia virus hasbeen used to incorporate an exogenous gene for expression of theexogenous gene product (see, for example, Perkus et al. Science229:981-984, 1985; Kaufman et al. Int. J. Cancer 48:900-907, 1991; MossScience 252:1662, 1991). A gene encoding an antigen of interest, such asan immunogenic Brachyury polypeptide, can be incorporated into the HINDF13L region or alternatively incorporated into the TK region ofrecombinant vaccinia virus vector (or other nonessential regions of thevaccinia virus genome). Baxby and Paoletti (Vaccine 10:8-9, 1992)disclose the construction and use as a vector, of the non-replicatingpoxvirus, including canarypox virus, fowlpox virus and other avianspecies. Sutter and Moss (Proc. Nat'l. Acad. Sci U.S.A. 89:10847-10851,1992) and Sutter et al. (Virology 1994) disclose the construction anduse as a vector, the non-replicating recombinant Ankara virus (MVA,modified vaccinia Ankara) in the construction and use of a vector.

Suitable vectors are disclosed, for example, in U.S. Pat. No. 6,998,252,which is incorporated herein by reference. In one example, a recombinantpoxvirus, such as a recombinant vaccinia virus is synthetically modifiedby insertion of a chimeric gene containing vaccinia regulatory sequencesor DNA sequences functionally equivalent thereto flanking DNA sequenceswhich in nature are not contiguous with the flanking vaccinia regulatoryDNA sequences that encode a Brachyury polypeptide. The recombinant viruscontaining such a chimeric gene is effective at expressing the Brachyurypolypeptide. In one example, the vaccine viral vector comprises (A) asegment comprised of (i) a first DNA sequence encoding a Brachyurypolypeptide and (ii) a poxvirus promoter, wherein the poxvirus promoteris adjacent to and exerts transcriptional control over the DNA sequenceencoding a Brachyury polypeptide; and, flanking said segment, (B) DNAfrom a nonessential region of a poxvirus genome. The viral vector canencode a selectable marker. In one example, the poxvirus includes, forexample, a thymidine kinase gene (see U.S. Pat. No. 6,998,252, which isincorporated herein by reference).

Poxviral vectors that encode a Brachyury polypeptide include at leastone expression control element operationally linked to the nucleic acidsequence encoding the Brachyury polypeptide. The expression controlelements are inserted in the poxviral vector to control and regulate theexpression of the nucleic acid sequence. Examples of expression controlelements of use in these vectors includes, but is not limited to, lacsystem, operator and promoter regions of phage lambda, yeast promotersand promoters derived from polyoma, adenovirus, retrovirus or SV40.Additional operational elements include, but are not limited to, leadersequence, termination codons, polyadenylation signals and any othersequences necessary for the appropriate transcription and subsequenttranslation of the nucleic acid sequence encoding the Brachyurypolypeptide in the host system. The expression vector can containadditional elements necessary for the transfer and subsequentreplication of the expression vector containing the nucleic acidsequence in the host system. Examples of such elements include, but arenot limited to, origins of replication and selectable markers. It willfurther be understood by one skilled in the art that such vectors areeasily constructed using conventional methods (Ausubel et al., (1987) in“Current Protocols in Molecular Biology,” John Wiley and Sons, New York,N.Y.) and are commercially available.

Basic techniques for preparing recombinant DNA viruses containing aheterologous DNA sequence encoding the Brachyury polypeptide, are knownin the art. Such techniques involve, for example, homologousrecombination between the viral DNA sequences flanking the DNA sequencein a donor plasmid and homologous sequences present in the parentalvirus (Mackett et al., 1982, Proc. Natl. Acad. Sci. USA 79:7415-7419).In particular, recombinant viral vectors such as a poxyviral vector canbe used in delivering the gene. The vector can be constructed forexample by steps known in the art, such as steps analogous to themethods for creating synthetic recombinants of the fowlpox virusdescribed in U.S. Pat. No. 5,093,258, incorporated herein by reference.Other techniques include using a unique restriction endonuclease sitethat is naturally present or artificially inserted in the parental viralvector to insert the heterologous DNA.

Generally, a DNA donor vector contains the following elements: (i) aprokaryotic origin of replication, so that the vector may be amplifiedin a prokaryotic host; (ii) a gene encoding a marker which allowsselection of prokaryotic host cells that contain the vector (e.g., agene encoding antibiotic resistance); (iii) at least one DNA sequenceencoding the Brachyury polypeptide located adjacent to a transcriptionalpromoter capable of directing the expression of the sequence; and (iv)DNA sequences homologous to the region of the parent virus genome wherethe foreign gene(s) will be inserted, flanking the construct of element(iii). Methods for constructing donor plasmids for the introduction ofmultiple foreign genes into pox virus are described in WO91/19803,incorporated herein by reference.

Generally, DNA fragments for construction of the donor vector, includingfragments containing transcriptional promoters and fragments containingsequences homologous to the region of the parent virus genome into whichforeign DNA sequences are to be inserted, can be obtained from genomicDNA or cloned DNA fragments. The donor plasmids can be mono, di-, ormultivalent (i.e., can contain one or more inserted foreign DNAsequences). The donor vector can contain an additional gene that encodesa marker that will allow identification of recombinant virusescontaining inserted foreign DNA. Several types of marker genes can beused to permit the identification and isolation of recombinant viruses.These include genes that encode antibiotic or chemical resistance (e.g.,see Spyropoulos et al., 1988, J. Virol. 62:1046; Falkner and Moss, 1988,J. Virol. 62:1849; Franke et al., 1985, Mol. Cell. Biol. 5:1918), aswell as genes such as the E. coli lacZ gene, that permit identificationof recombinant viral plaques by colorimetric assay (Panicali et al.,1986, Gene 47:193-199).

The DNA gene sequence to be inserted into the virus can be placed into adonor plasmid, such as an E. coli or a Salmonella plasmid construct,into which DNA homologous to a section of DNA such as that of theinsertion site of the poxvirus where the DNA is to be inserted has beeninserted. Separately the DNA gene sequence to be inserted is ligated toa promoter. The promoter-gene linkage is positioned in the plasmidconstruct so that the promoter-gene linkage is flanked on both ends byDNA homologous to a DNA sequence flanking a region of pox DNA that isthe desired insertion region. With a parental pox viral vector, a poxpromoter is used. The resulting plasmid construct is then amplified bygrowth within E. coli bacteria and isolated. Next, the isolated plasmidcontaining the DNA gene sequence to be inserted is transfected into acell culture, for example chick embryo fibroblasts, along with theparental virus, for example poxvirus. Recombination between homologouspox DNA in the plasmid and the viral genome respectively results in arecombinant poxvirus modified by the presence of the promoter-geneconstruct in its genome, at a site that does not affect virus viability.

As noted above, the DNA sequence is inserted into a region (insertionregion) in the virus that does not affect virus viability of theresultant recombinant virus. One of skill in the art can readilyidentify such regions in a virus by, for example, randomly testingsegments of virus DNA for regions that allow recombinant formationwithout seriously affecting virus viability of the recombinant. Oneregion that can readily be used and is present in many viruses is thethymidine kinase (TK) gene. The TK gene has been found in all pox virusgenomes examined, including leporipoxvirus (Upton et al., 1986, J.Virology 60:920); shope fibromavirus; capripoxvirus (Gershon et al.,1989, J. Gen. Virol. 70:525) Kenya sheep-1; orthopoxvirus (Weir et al.,1983, J. Virol. 46:530) vaccinia (Esposito et al., 1984, Virology135:561); monkeypox and variola virus (Hruby et al., 1983, PNAS 80:3411)vaccinia (Kilpatrick et al., 1985, Virology 143:399); Yaba monkey tumorvirus; avipoxvirus (Binns et al., 1988, J. Gen. Virol. 69:1275);fowipox; (Boyle et al., 1987, Virology 156:355); fowlpox (Schnitzlein etal., 1988, J. Virological Methods 20:341); fowlpox, quailpox; entomopox(Lytvyn et al., 1992, J. Gen. Virol. 73:3235-3240). In vaccinia, inaddition to the TK region, other insertion regions include, for example,the HindIII fragment. In fowlpox, in addition to the TK region, otherinsertion regions include, for example, the BamHI J fragment (Jenkins etal., 1991, AIDS Research and Human Retroviruses 7:991-998) theECORI-HindIII fragment, EcoRV-HindIII fragment, BamHI fragment and theHindIII fragment set forth in EPO Application No. 0 308220 A1 (see alsoCalvert et al., 1993, J. Virol. 67:3069-3076; Taylor et al., 1988,Vaccine 6:497-503; Spehner et al., 1990; Boursnell et al., 1990, J. Gen.Virol. 71:621-628).

In swinepox, insertion sites include the thymidine kinase gene region.In addition to the requirement that the gene be inserted into aninsertion region, successful expression of the inserted gene by themodified poxvirus requires the presence of a promoter operably linked tothe desired gene. Generally, the promoter is placed so that it islocated upstream from the gene to be expressed. Promoters are well knownin the art and can readily be selected depending on the host and thecell type you wish to target. In one example, in poxviruses, pox viralpromoters are used, such as the vaccinia 7.5K, 40K or fowlpox promoterssuch as FPV CIA. Enhancer elements can also be used in combination toincrease the level of expression. Furthermore, inducible promoters canbe utilized.

Homologous recombination between donor plasmid DNA and viral DNA in aninfected cell can result in the formation of recombinant viruses thatincorporate the desired elements. Appropriate host cells for in vivorecombination are generally eukaryotic cells that can be infected by thevirus and transfected by the plasmid vector. Examples of such cellssuitable for use with a pox virus are chick embryo fibroblasts, HuTK143(human) cells, and CV-1 and BSC-40 (both monkey kidney) cells. Infectionof cells with pox virus and transfection of these cells with plasmidvectors is accomplished by techniques standard in the art (see U.S. Pat.No. 4,603,112 and PCT Publication No. WO 89/03429).

Following in vivo recombination, recombinant viral progeny can beidentified by one of several techniques. For example, if the DNA donorvector is designed to insert foreign genes into the parent virusthymidine kinase (TK) gene, viruses containing integrated DNA will beTK- and can be selected on this basis (Mackett et al., 1982, Proc. Natl.Acad. Sci. USA 79:7415). Alternatively, co-integration of a geneencoding a marker or indicator gene with the foreign gene(s) ofinterest, as described above, can be used to identify recombinantprogeny. One specific non-limiting example of an indicator gene is theE. coli lacZ gene. Recombinant viruses expressing beta-galactosidase canbe selected using a chromogenic substrate for the enzyme (Panicali etal., 1986, Gene 47:193). Once a recombinant virus has been identified, avariety of well-known methods can be used to assay the expression of theBrachyury sequence encoded by the inserted DNA fragment. These methodsinclude black plaque assay (an in situ enzyme immunoassay performed onviral plaques), Western blot analysis, radioimmunoprecipitation (RIPA),and enzyme immunoassay (EIA).

This disclosure encompasses a recombinant virus comprising more than oneantigen of interest for the purpose of having a multivalent vaccine. Forexample, the recombinant virus may comprise the virus genome or portionsthereof, the nucleic acid sequence encoding the Brachyury polypeptideand a nucleic acid sequence encoding a hepatitis B surface antigen.

In one embodiment, a composition is provided that includes a recombinantvirus comprising a vaccinia virus genome or portions thereof, thenucleic acid sequence encoding a Brachyury polypeptide and a recombinantvirus comprising the nucleic acid sequence encoding theimmunostimulatory molecule, B 7.1 alone or in combination with thenucleic acid sequence encoding the immunostimulatory molecule, B7-2, ora recombinant virus containing both the genes for a tumor antigen and animmunostimulatory molecule. This disclosure also encompasses arecombinant virus comprising the Brachyury polypeptide that isadministered with a second recombinant virus comprising the virus genomeor portion thereof, and one or more nucleic acid sequences encoding oneor more B7 molecules, such as a recombinant vaccinia virus expressingB7-1 and/or B7-2. It is disclosed in U.S. Pat. No. 893,869 (incorporatedby reference herein) that the rapid infection of tumor cells with theserecombinant viruses demonstrates that vaccinia can authentically expressthese proteins and that they are functional molecules. Followingtransfer of the nucleic acids, weakly immunogenic syngeneic tumorsexpressing these recombinant molecules are rejected by immunocompetenthosts.

Thus, in one example, recombinant virus is disclosed that is arecombinant vaccinia virus containing B7-1 and a recombinant vacciniavirus containing B7-2 (designated rV-B7-1 and rV-B7-2, respectively);the composition can include rV-B7-1 and/or rV-B7-2 in combination withan immunogenic Brachyury polypeptide.

The B7 molecule includes but is not limited to B7-1, B7-2 and analogsthereof. The B7 gene may be cloned from mammalian sources, including butnot limited to mammalian tissues, genomic libraries or cDNA libraries,such as from murine or human sources. Without being bound by theory,co-stimulatory molecules of the B7 family (namely B7-1, B7-2, andpossibly B7.3) are believed to be members of the immunoglobulin genesuperfamily. These molecules are present on macrophages, dendriticcells, monocytes (antigen presenting cells (APCs)). Significantamplification of the immune response against a given antigen generallydoes not occur without co-stimulation (June et al. (Immunology Today15:321-331, 1994); Chen et al. (Immunology Today 14:483-486); Townsendet al. (Science 259:368-370)). Freeman et al. (J. Immunol.143:2714-2722, 1989) report cloning and sequencing of B7-1 gene. Azumaet al. Nature 366:76-79, 1993) report cloning and sequencing B7-2 gene.Thus, in one embodiment the B7-1 gene or the B7-2 genes are administeredin conjunction with the Brachyury polypeptide. The insertion of nucleicacids encoding B7-1 and B7-2 into vaccinia virus has been disclosed (seefor example, U.S. Pat. No. 6,893,869, incorporated herein by reference;this U.S. Patent also discloses the use of a nucleic acid encoding IL-2in a vaccinia virus). Several vectors including IL-2, B7-1 and B7-2 havebeen deposited with the American Type Culture Collection (ATCC) on Oct.3, 1994 under the terms of the Budapest Treaty (for example,rV-CEA/_(n)IL-2 (ATCC Designation VR 2480), rV-_(m)B7-2 (ATCCDesignation VR 2482); and rV-_(m)B7-1 (ATCC Designation VR 2483)).

DNA sequences encoding a Brachyury polypeptide can be expressed in vitroby DNA transfer into a suitable host cell. The cell may be prokaryoticor eukaryotic. The term also includes any progeny of the subject hostcell. It is understood that all progeny may not be identical to theparental cell since there may be mutations that occur duringreplication. Methods of stable transfer, meaning that the foreign DNA iscontinuously maintained in the host, are known in the art.

As noted above, a polynucleotide sequence encoding a Brachyurypolypeptide can be operatively linked to expression control sequences.An expression control sequence operatively linked to a coding sequenceis ligated such that expression of the coding sequence is achieved underconditions compatible with the expression control sequences. Theexpression control sequences include, but are not limited to,appropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene toperrrrit proper translation of mRNA, and stop codons.

Hosts cells can include microbial, yeast, insect and mammalian hostcells. Methods of expressing DNA sequences having eukaryotic or viralsequences in prokaryotes are well known in the art. Non-limitingexamples of suitable host cells include bacteria, archea, insect, fungi(for example, yeast), plant, and animal cells (for example, mammaliancells, such as human). Exemplary cells of use include Escherichia coli,Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9cells, C129 cells, 293 cells, Neurospora, and immortalized mammalianmyeloid and lymphoid cell lines. Techniques for the propagation ofmammalian cells in culture are well-known (see, Jakoby and Pastan (eds),1979, Cell Culture. Methods in Enzymology, volume 58, Academic Press,Inc., Harcourt Brace Jovanovich, N.Y.). Examples of commonly usedmammalian host cell lines are VERO and HeLa cells, CHO cells, and WI38,BHK, and COS cell lines, although cell lines may be used, such as cellsdesigned to provide higher expression desirable glycosylation patterns,or other features. As discussed above, techniques for the transformationof yeast cells, such as polyethylene glycol transformation, protoplasttransformation and gene guns are also known in the art (see Gietz andWoods Methods in Enzymology 350: 87-96, 2002).

Transformation of a host cell with recombinant DNA can be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as, but not limited to, E. coli,competent cells which are capable of DNA uptake can be prepared fromcells harvested after exponential growth phase and subsequently treatedby the CaCl₂ method using procedures well known in the art.Alternatively, MgCl₂ or RbCl can be used. Transformation can also beperformed after forming a protoplast of the host cell if desired, or byelectroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors can be used. Eukaryotic cells can also beco-transformed with polynucleotide sequences encoding a Brachyurypolypeptide, and a second foreign DNA molecule encoding a selectablephenotype, such as the herpes simplex thymidine kinase gene. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein (see for example, EukaryoticViral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

Therapeutic Methods and Pharmaceutical Compositions

The Brachyury polypeptides disclosed herein, or nucleic acids encodingthe Brachyury polypeptides, can be used to generate an immune responsein a subject. In several examples, the subject has a tumor thatexpresses Brachyury. Thus, in several embodiments, the methods includeadministering to a subject with cancer a therapeutically effectiveamount of one or more of the Brachyury polypeptides disclosed herein, inorder to generate an immune response.

The methods can include selecting a subject in need of treatment, suchas a subject with a tumor that expresses Brachyury. In several examples,the methods include selecting a subject with a tumor of the smallintestine, stomach, kidney, bladder, uterus, ovaries, testes lung, colonor prostate. In additional examples, the method includes selecting asubject with a tumor of B cell origin, such as chronic lymphocyticleukemia (CLL), a B cell lymphoma, Burkitt's lymphoma or a Hodgkin'slymphoma.

In exemplary applications, compositions are administered to a subjecthaving a disease, such as cancer (for example, small intestine, stomach,kidney, bladder, uterus, ovary, testes, lung colon, or prostate cancer),in an amount sufficient to raise an immune response toBrachyury-expressing cells. Administration induces a sufficient immuneresponse to slow the proliferation of such cells or to inhibit theirgrowth, or to reduce a sign or a symptom of the tumor. Amounts effectivefor this use will depend upon the severity of the disease, the generalstate of the patient's health, and the robustness of the patient'simmune system. In one example, a therapeutically effective amount of thecompound is that which provides either subjective relief of a symptom(s)or an objectively identifiable improvement as noted by the clinician orother qualified observer.

A Brachyury polypeptide can be administered by any means known to one ofskill in the art (see Banga, A., “Parenteral Controlled Delivery ofTherapeutic Peptides and Proteins,” in Therapeutic Peptides andProteins, Technomic Publishing Co., Inc., Lancaster, Pa., 1995) eitherlocally or systemically, such as by intramuscular, subcutaneous,intraperitoneal or intravenous injection, but even oral, nasal,transdermal or anal administration is contemplated. In one embodiment,administration is by subcutaneous or intramuscular injection. To extendthe time during which the peptide or protein is available to stimulate aresponse, the peptide or protein can be provided as an implant, an oilyinjection, or as a particulate system. The particulate system can be amicroparticle, a microcapsule, a microsphere, a nanocapsule, or similarparticle. (see, e.g., Banga, supra). A particulate carrier based on asynthetic polymer has been shown to act as an adjuvant to enhance theimmune response, in addition to providing a controlled release. Aluminumsalts can also be used as adjuvants to produce an immune response.

In one specific, non-limiting example, the Brachyury polypeptide isadministered in a manner to direct the immune response to a cellularresponse (that is, a cytotoxic T lymphocyte (CTL) response), rather thana humoral (antibody) response.

Optionally, one or more cytokines, such as IL-2, IL-6, IL-12, RANTES,GM-CSF, TNF-α, or IFN-γ, one or more growth factors, such as GM-CSF orG-CSF, one or more costimulatory molecules, such as ICAM-1, LFA-3, CD72,B7-1, B7-2, or other B7 related molecules; one or more molecules such asOX-40L or 41 BBL, or combinations of these molecules, can be used asbiological adjuvants (see, for example, Salgaller et al., 1998, J. Surg.Oncol. 68(2):122-38; Lotze et al., 2000, Cancer J Sci. Am. 6(Suppl1):S61-6; Cao et al., 1998, Stem Cells 16(Suppl 1):251-60; Kuiper etal., 2000, Adv. Exp. Med. Biol. 465:381-90). These molecules can beadministered systemically (or locally) to the host. In several examples,IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, B7-1B7-2, OX-40L, 41 BBL and ICAM-1 are administered.

A number of means for inducing cellular responses, both in vitro and invivo, are known. Lipids have been identified as agents capable ofassisting in priming CTL in vivo against various antigens. For example,as described in U.S. Pat. No. 5,662,907, palmitic acid residues can beattached to the alpha and epsilon amino groups of a lysine residue andthen linked (for example, via one or more linking residues, such asglycine, glycine-glycine, serine, serine-serine, or the like) to animmunogenic peptide. The lipidated peptide can then be injected directlyin a micellar form, incorporated in a liposome, or emulsified in anadjuvant. As another example, E. coli lipoproteins, such astripalmitoyl-S-glycerylcysteinlyseryl-serine can be used to prime tumorspecific CTL when covalently attached to an appropriate peptide (see,Deres et al., Nature 342:561, 1989). Further, as the induction ofneutralizing antibodies can also be primed with the same moleculeconjugated to a peptide which displays an appropriate epitope, twocompositions can be combined to elicit both humoral and cell-mediatedresponses where that is deemed desirable.

In yet another embodiment, to induce a CTL response to an immunogenicBrachyury polypeptide, a MHC Class II-restricted T-helper epitope isadded to the immunogenic Brachyury polypeptide to induce T-helper cellsto secrete cytokines in the microenvironment to activate CTL precursorcells. The technique further involves adding short lipid molecules toretain the construct at the site of the injection for several days tolocalize the antigen at the site of the injection and enhance itsproximity to dendritic cells or other “professional” antigen presentingcells over a period of time (see Chesnut et al., “Design and Testing ofPeptide-Based Cytotoxic T-Cell-Mediated Immunotherapeutics to TreatInfectious Diseases and Cancer,” in Powell et al., eds., Vaccine Design,the Subunit and Adjuvant Approach, Plenum Press, New York, 1995).

A pharmaceutical composition including a Brachyury polypeptide is thusprovided. These compositions are use to generate an immune response,such as for immunotherapy. In one embodiment, the Brachyury polypeptideis mixed with an adjuvant containing two or more of a stabilizingdetergent, a micelle-forming agent, and an oil. Suitable stabilizingdetergents, micelle-forming agents, and oils are detailed in U.S. Pat.No. 5,585,103; U.S. Pat. No. 5,709,860; U.S. Pat. No. 5,270,202; andU.S. Pat. No. 5,695,770, all of which are incorporated by reference. Astabilizing detergent is any detergent that allows the components of theemulsion to remain as a stable emulsion. Such detergents includepolysorbate, 80 (TWEEN)(Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl; manufactured byICI Americas, Wilmington, Del.), TWEEN 40™, TWEEN 20™, TWEEN 60™Zwittergent™ 3-12, TEEPOL HB7™, and SPAN 85™. These detergents areusually provided in an amount of approximately 0.05 to 0.5%, such as atabout 0.2%.

A micelle forming agent is an agent which is able to stabilize theemulsion formed with the other components such that a micelle-likestructure is formed. Such agents generally cause some irritation at thesite of injection in order to recruit macrophages to enhance thecellular response. Examples of such agents include polymer surfactantsdescribed by BASF Wyandotte publications, e.g., Schmolka, J. Am. Oil.Chem. Soc. 54:110, 1977, and Hunter et al., J. Immunol 129:1244, 1981,PLURONIC™ L62LF, L101, and L64, PEG1000, and TETRONIC™ 1501, 150R1, 701,901, 1301, and 130R1. The chemical structures of such agents are wellknown in the art. In one embodiment, the agent is chosen to have ahydrophile-lipophile balance (HLB) of between 0 and 2, as defined byHunter and Bennett, J. Immun. 133:3167, 1984. The agent can be providedin an effective amount, for example between 0.5 and 10%, or in an amountbetween 1.25 and 5%.

The oil included in the composition is chosen to promote the retentionof the antigen in oil-in-water emulsion, such as to provide a vehiclefor the desired antigen, and preferably has a melting temperature ofless than 65° C. such that emulsion is formed either at room temperature(about 20° C. to 25° C.), or once the temperature of the emulsion isbrought down to room temperature. Examples of such oils includesqualene, Squalane, EICOSANE™, tetratetracontane, glycerol, and peanutoil or other vegetable oils. In one specific, non-limiting example, theoil is provided in an amount between 1 and 10%, or between 2.5 and 5%.The oil should be both biodegradable and biocompatible so that the bodycan break down the oil over time, and so that no adverse affects, suchas granulomas, are evident upon use of the oil.

In one embodiment, the adjuvant is a mixture of stabilizing detergents,micelle-forming agent, and oil available under the name PROVAX® (IDECPharmaceuticals, San Diego, Calif.). An adjuvant can also be animmunostimulatory nucleic acid, such as a nucleic acid including a CpGmotif, or a biological adjuvant (see above).

Controlled release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems, see Banga, Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., 1995. Particulate systems includemicrospheres, microparticles, microcapsules, nanocapsules, nanospheres,and nanoparticles. Microcapsules contain the therapeutic protein as acentral core. In microspheres, the therapeutic agent is dispersedthroughout the particle. Particles, microspheres, and microcapsulessmaller than about 1 μm are generally referred to as nanoparticles,nanospheres, and nanocapsules, respectively. Capillaries have a diameterof approximately 5 μm so that only nanoparticles are administeredintravenously. Microparticles are typically around 100 μm in diameterand are administered subcutaneously or intramuscularly (see Kreuter,Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc.,New York, N.Y., pp. 219-342, 1994; Tice & Tabibi, Treatise on ControlledDrug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y.,pp. 315-339, 1992).

Polymers can be used for ion-controlled release. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, Accounts Chem. Res. 26:537, 1993). Forexample, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston etal., Pharm. Res. 9:425, 1992; and Pec, J. Parent. Sci. Tech. 44(2):58,1990). Alternatively, hydroxyapatite has been used as a microcarrier forcontrolled release of proteins (Ijntema et al., Int. J. Pharm. 112:215,1994). In yet another aspect, liposomes are used for controlled releaseas well as drug targeting of the lipid-capsulated drug (Betageri et al.,Liposome Drug Delivery Systems, Technomic Publishing Co., Inc.,Lancaster, Pa., 1993). Numerous additional systems for controlleddelivery of therapeutic proteins are known (e.g., U.S. Pat. No.5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No. 4,235,871; U.S. Pat.No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat. No. 4,957,735; andU.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S. Pat. No.5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164; U.S. Pat.No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No. 5,506,206; U.S.Pat. No. 5,271,961; U.S. Pat. No. 5,254,342; and U.S. Pat. No.5,534,496).

In another embodiment, a pharmaceutical composition includes a nucleicacid encoding an Brachyury polypeptide. A therapeutically effectiveamount of the Brachyury polynucleotide can be administered to a subjectin order to generate an immune response. In one specific, non-limitingexample, a therapeutically effective amount of the Brachyurypolynucleotide is administered to a subject to treat prostate cancer orbreast cancer.

Optionally, one or more cytokines, such as IL-2, IL-6, IL-12, RANTES,GM-CSF, TNF-α, or IFN-γ, one or more growth factors, such as GM-CSF orG-CSF, one or more costimulatory molecules, such as ICAM-1, LFA-3, CD72,B7-1, B7-2, or other B7 related molecules; one or more molecules such asOX-40L or 41 BBL, or combinations of these molecules, can be used asbiological adjuvants (see, for example, Salgaller et al., 1998, J. Surg.Oncol. 68(2):122-38; Lotze et al., 2000, Cancer J Sci. Am. 6(Suppl1):S61-6; Cao et al., 1998, Stem Cells 16(Suppl 1):251-60; Kuiper etal., 2000, Adv. Exp. Med. Biol. 465:381-90). These molecules can beadministered systemically to the host. It should be noted that thesemolecules can be co-administered via insertion of a nucleic acidencoding the molecules into a vector, for example, a recombinant poxvector (see, for example, U.S. Pat. No. 6,045,802). In variousembodiments, the nucleic acid encoding the biological adjuvant can becloned into same vector as the Brachyury polypeptide coding sequence, orthe nucleic acid can be cloned into one or more separate vectors forco-administration. In addition, nonspecific immunomodulating factorssuch as Bacillus Cahnette-Guerin (BCG) and levamisole can beco-administered.

One approach to administration of nucleic acids is direct immunizationwith plasmid DNA, such as with a mammalian expression plasmid. Asdescribed above, the nucleotide sequence encoding a Brachyurypolypeptide can be placed under the control of a promoter to increaseexpression of the molecule.

Immunization by nucleic acid constructs is well known in the art andtaught, for example, in U.S. Pat. No. 5,643,578 (which describes methodsof immunizing vertebrates by introducing DNA encoding a desired antigento elicit a cell-mediated or a humoral response), and U.S. Pat. No.5,593,972 and U.S. Pat. No. 5,817,637 (which describe operably linking anucleic acid sequence encoding an antigen to regulatory sequencesenabling expression). U.S. Pat. No. 5,880,103 describes several methodsof delivery of nucleic acids encoding immunogenic peptides or otherantigens to an organism. The methods include liposomal delivery of thenucleic acids (or of the synthetic peptides themselves), andimmune-stimulating constructs, or ISCOMS™, negatively charged cage-likestructures of 30-40 nm in size formed spontaneously on mixingcholesterol and Quil A™ (saponin). Protective immunity has beengenerated in a variety of experimental models of infection, includingtoxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMS™ asthe delivery vehicle for antigens (Mowat and Donachie, Immunol. Today12:383, 1991). Doses of antigen as low as 1 μg encapsulated in ISCOMS™have been found to produce Class I mediated CTL responses (Takahashi etal., Nature 344:873, 1990).

In another approach to using nucleic acids for immunization, a Brachyurypolypeptide can also be expressed by attenuated viral hosts or vectorsor bacterial vectors. Recombinant vaccinia virus, adeno-associated virus(AAV), herpes virus, retrovirus, or other viral vectors can be used toexpress the peptide or protein, thereby eliciting a CTL response. Forexample, vaccinia vectors and methods useful in immunization protocolsare described in U.S. Pat. No. 4,722,848. BCG (Bacillus Calmette Guerin)provides another vector for expression of the peptides (see Stover,Nature 351:456-460, 1991).

A first recombinant virus, such as a poxvirus (for example, vaccinevirus) encoding a Brachyury immunogenic polypeptide can be used inconjunction with a second recombinant virus which has incorporated intoa viral genome or infectable portion thereof one or more genes or DNAsequences encoding B7-1, B7-2, or B7-1 and B7-2, wherein the compositionis able to coinfect a host cell resulting in coexpression of thepolypeptide and the B7-1, B7-2, or B7-1 and B7-2 encoding genes or DNAsequences (see U.S. Pat. No. 6,893,869, and U.S. Pat. No. 6,045,908,which are incorporated by reference herein). The expression of the B7gene family has been shown to be an important mechanism of anti-tumorresponses in both mice and humans.

When a viral vector is utilized, it is desirable to provide therecipient with a dosage of each recombinant virus in the composition inthe range of from about 10⁵ to about 10¹⁰ plaque forming units/mgmammal, although a lower or higher dose can be administered. Thecomposition of recombinant viral vectors can be introduced into a mammaleither prior to any evidence of a cancer, or to mediate regression ofthe disease in a mammal afflicted with the cancer. Examples of methodsfor administering the composition into mammals include, but are notlimited to, exposure of cells to the recombinant virus ex vivo, orinjection of the composition into the affected tissue or intravenous,subcutaneous, intradermal or intramuscular administration of the virus.Alternatively the recombinant viral vector or combination of recombinantviral vectors may be administered locally by direct injection into thecancerous lesion in a pharmaceutically acceptable carrier. Generally,the quantity of recombinant viral vector, carrying the nucleic acidsequence of one or more Brachyury polypeptides to be administered isbased on the titer of virus particles. An exemplary range of theimmunogen to be administered is 10⁵ to 10¹⁰ virus particles per mammal,such as a human.

In the embodiment where a combination of a first recombinant viralvector carrying a nucleic acid sequence of one or more Brachyurypolypeptides and a second recombinant viral vector carrying the nucleicacid sequence of one or more immunostimulatory molecules is used, themammal can be immunized with different ratios of the first and secondrecombinant viral vector. In one embodiment the ratio of the firstvector to the second vector is about 1:1, or about 1:3, or about 1:5.Optimal ratios of the first vector to the second vector may easily betitered using the methods known in the art (see, for example, U.S. Pat.No. 6,893,869, incorporated herein by reference).

In one embodiment the recombinant viruses have been constructed toexpress cytokines (such as TNF-α, IL-6, GM-CSF, and IL-2), andco-stimulatory and accessory molecules (B7-1, B7-2) alone and in avariety of combinations. Simultaneous production of an immunostimulatorymolecule and the Brachyury polypeptide enhances the generation ofspecific effectors. Without being bound by theory, dependent upon thespecific immunostimulatory molecules, different mechanisms might beresponsible for the enhanced immunogenicity: augmentation of help signal(IL-2), recruitment of professional APC (GM-CSF), increase in CTLfrequency (IL-2), effect on antigen processing pathway and MHCexpression (IFNγ and TNFα) and the like. For example, IL-2, IL-6,interferon, tumor necrosis factor, or a nucleic acid encoding thesemolecules, can be administered in conjunction with a Brachyuryimmunogenic polypeptide, or a nucleic acid encoding a Brachyurypolypeptide. The co-expression of a Brachyury polypeptide together withat least one immunostimulatory molecule can be effective in an animalmodel to show anti-tumor effects. In one embodiment, a nucleic acidencoding a Brachyury polypeptide is introduced directly into cells. Forexample, the nucleic acid can be loaded onto gold microspheres bystandard methods and introduced into the skin by a device such asBio-Rad's Helios™ Gene Gun. The nucleic acids can be “naked,” consistingof plasmids under control of a strong promoter. Typically, the DNA isinjected into muscle, although it can also be injected directly intoother sites, including tissues in proximity to metastases. Dosages forinjection are usually around 0.5 μg/kg to about 50 mg/kg, and typicallyare about 0.005 mg/kg to about 5 mg/kg (see, for example, U.S. Pat. No.5,589,466).

In one specific, non-limiting example, a pharmaceutical composition forintravenous administration would include about 0.1 μg to 10 mg ofimmunogenic Brachyury polypeptide per patient per day. Dosages from 0.1up to about 100 mg per patient per day can be used, particularly if theagent is administered to a secluded site and not into the circulatory orlymph system, such as into a body cavity or into a lumen of an organ.Actual methods for preparing administrable compositions will be known orapparent to those skilled in the art and are described in more detail insuch publications as Remingtons Pharmaceutical Sciences, 19^(th) Ed.,Mack Publishing Company, Easton, Pa., 1995.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thesubject. In one embodiment, the dosage is administered once as a bolus,but in another embodiment can be applied periodically until atherapeutic result is achieved. Generally, the dose is sufficient totreat or ameliorate symptoms or signs of disease without producingunacceptable toxicity to the subject. Systemic or local administrationcan be utilized.

In another method, antigen presenting cells (APCs), such as dendriticcells, are pulsed or co-incubated with peptides comprising a Brachyurypolypeptide in vitro. In one specific, non-limiting example, the antigenpresenting cells can be autologous cells. A therapeutically effectiveamount of the antigen presenting cells can then be administered to asubject.

The Brachyury polypeptide can be delivered to the dendritic cells or todendritic cell precursors via any method known in the art, including,but not limited to, pulsing dendritic cells directly with antigen, orutilizing a broad variety of antigen delivery vehicles, such as, forexample, liposomes, or other vectors known to deliver antigen to cells.In one specific, non-limiting example an antigenic formulation includesabout 0.1 μg to about 1,000 μg, or about 1 to about 100 μg of a selectedBrachyury polypeptide. The Brachyury polypeptide can also beadministered with agents that promote dendritic cell maturation.Specific, non-limiting examples of agents of use are interleukin-4(IL-4) and granulocyte/macrophage colony stimulating factor (GM-CSF), orflt-3 ligand (flt-3L). The preparation can also contain buffers,excipients, and preservatives, amongst other ingredients.

In one embodiment, mature antigen presenting cells are generated topresent the immunogenic Brachyury polypeptide. These dendritic cells arethen administered alone (or in combination with another agent) to asubject with a tumor that expresses Brachyury, such as a smallintestine, stomach, kidney, bladder, uterus, ovary, testis, lung colonand/or prostate cancer. The dendritic cells can also be administered toa subject with a tumor of B cell origin, such as chronic lymphocyticleukemia (CLL), a B cell lymphoma, Burkitt's lymphoma or a Hodgkin'slymphoma.

In another embodiment, the mature dendritic cells are administered inconjunction with a chemotherapeutic agent.

Alternatively, the APCs are used to sensitize CD8 cells, such as tumorinfiltrating lymphocytes (TILs) from tumors or peripheral bloodlymphocytes (PBLs). The TILs or PBLs can be from the same subject(autologous) that is to be treated. Alternatively, the TILs or PBLs canbe heterologous. However, they should at least be MHC Class-I restrictedto the HLA types the subject possesses. An effective amount of thesensitized cells are then administered to the subject.

Peripheral blood mononuclear cells (PBMCs) can be used as the respondercell source of CTL precursors. The appropriate antigen-presenting cellsare incubated with peptide, after which the peptide-loadedantigen-presenting cells are then incubated with the responder cellpopulation under optimized culture conditions. Positive CTL activationcan be determined by assaying the culture for the presence of CTLs thatkill radio-labeled target cells, both specific peptide-pulsed targets aswell as target cells expressing endogenously processed forms of theantigen from which the peptide sequence was derived, such as Brachyury(for example, SEQ ID NO: 1).

The cells can be administered to a subject to inhibit the growth ofcells of Brachyury expressing tumors. In these applications, atherapeutically effective amount of activated antigen presenting cells,or activated lymphocytes, are administered to a subject suffering from adisease, in an amount sufficient to raise an immune response toBrachyury-expressing cells. The resulting immune response is sufficientto slow the proliferation of such cells or to inhibit their growth, orto reduce a sign or a symptom of the tumor.

In a supplemental method, any of these immunotherapies is augmented byadministering a cytokine, such as interleukin (IL)-2, IL-3, IL-6, IL-10,IL-12, IL-15, GM-CSF, or interferons.

In a further method, any of these immunotherapies is augmented byadministering an additional chemotherapeutic agent. In one example, thisadministration is sequential. Examples of such agents are alkylatingagents, antimetabolites, natural products, or hormones and theirantagonists. Examples of alkylating agents include nitrogen mustards(such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard orchlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (suchas carmustine, lomustine, semustine, streptozocin, or dacarbazine).Examples of antimetabolites include folic acid analogs (such asmethotrexate), pyrimidine analogs (such as 5-FU or cytarabine), andpurine analogs, such as mercaptopurine or thioguanine. Examples ofnatural products include vinca alkaloids (such as vinblastine,vincristine, or vindesine), epipodophyllotoxins (such as etoposide orteniposide), antibiotics (such as dactinomycin, daunorubicin,doxorubicin, bleomycin, plicamycin, or mitocycin C), and enzymes (suchas L-asparaginase). Examples of miscellaneous agents include platinumcoordination complexes (such as cis-diamine-dichloroplatinum II alsoknown as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), and adrenocroticalsuppressants (such as mitotane and aminoglutethimide). Examples ofhormones and antagonists include adrenocorticosteroids (such asprednisone), progestins (such as hydroxyprogesterone caproate,medroxyprogesterone acetate, and magestrol acetate), estrogens (such asdiethylstilbestrol and ethinyl estradiol), antiestrogens (such astamoxifen), and androgens (such as testosterone proprionate andfluoxymesterone). Examples of the most commonly used chemotherapy drugsthat can be concurrently administered with the disclosed immunotherapyinclude Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU,Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU,Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin,Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, suchas docetaxel), Velban, Vincristine, VP-16, while some more newer drugsinclude Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11),Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin),Xeloda (Capecitabine), Zevelin and calcitriol. Non-limiting examples ofimmunomodulators that can be used include AS-101 (Wyeth-Ayerst Labs.),bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocytemacrophage colony stimulating factor; Genetics Institute), IL-2 (Cetusor Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG(from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosisfactor; Genentech).

Methods of Treatment Using Specific Binding Agents

Expression of Brachyury is associated with tumor cell migration andinvasion. Brachyury is expressed in small intestine, stomach, kidney,bladder, uterus, ovaries, testes lung, colon and prostate tumors but notin most normal tissues. In addition, expression of Brachyury isassociated with epithelial-to mesenchymal transition. Moreover,Brachyury is expressed in tumors of B cell origin, such as chroniclymphocytic leukemia (CLL), Epstein-Barr virus transformed B cells,Burkitt's and Hodgkin's lymphomas. Reagents that can reduce theexpression of Brachyury also can be used to treat tumors that expressBrachyury. The reagents can be used alone, or can be used in combinationwith the immunogenic Brachyury polypeptides disclosed herein.

The methods can include selecting a subject in need of treatment, suchas a subject with a tumor that expresses Brachyury. In several examples,the methods include selecting a subject with a tumor of the smallintestine, stomach, kidney, bladder, uterus, ovaries, testes lung, colonor prostate. In additional examples, the method includes selecting asubject with a tumor of B cell origin, such as chronic lymphocyticleukemia (CLL), a B cell lymphoma, Burkitt's lymphoma or a Hodgkin'slymphoma.

In one example, the method includes administering a therapeuticallyeffective amount of a specific binding agent that preferentially bindsto Brachyury. The specific binding agent can be an inhibitor such as asiRNA or an antisense molecule that specifically binds Brachyury mRNA(such as an mRNA encoding SEQ ID NO: 1). Inhibition of Brachyury doesnot require 100% inhibition, but can include at least a reduction if nota complete inhibition of cell growth or differentiation associated witha specific pathological condition. Treatment of a tumor by reducingBrachyury expression can include delaying the development of the tumorin a subject (such as preventing metastasis of a tumor) by altering theability of the tumor to metastasize. Treatment of a tumor also includesreducing signs or symptoms associated with the presence of such a tumor(for example by reducing the size or volume of the tumor or a metastasisthereof) by decreasing the number of metastases. In some examplesdecrease or slowing metastasis of the tumor, or reducing the size orvolume of the tumor, is an alteration of at least 10%, at least 20%, atleast 50%, or at least 75. In some examples, treatment using the methodsdisclosed herein prolongs the time of survival of the subject. Treatmentcan also result in a down-regulation of mesenchymal markers (such asfibronectin, vimentin and/or N-cadherin) and an up-regulation ofepithelial markers (such as E-cadherin or g-catenin).

Specific binding agents are agents that bind with higher affinity toBrachyury, than to other molecules. For example, a specific bindingagent can be one that binds with high affinity to Brachyury but does notsubstantially bind to another gene or gene product. For example, thespecific binding agent interferes with gene expression (transcription,processing, translation, post-translational modification), such as, byinterfering with Brachyury mRNA and blocking translation into protein.

A reduction of Brachyury protein expression in a target cell may beobtained by introducing into cells an antisense or other suppressiveconstruct based on the Brachyury coding sequence. For antisensesuppression, a nucleotide sequence from a Brachyury encoding sequence,e.g. all or a portion of the Brachyury cDNA or gene, is arranged inreverse orientation relative to the promoter sequence in thetransformation vector.

The introduced sequence need not be the full length Brachyury gene, andneed not be exactly homologous to the equivalent sequence found in thecell type to be transformed. Thus, portions or fragments of a nucleicacid encoding Brachyury (SEQ ID NO: 2) could also be used to knock outor suppress expression. Generally, however, where the introducedsequence is of shorter length, a higher degree of identity to the nativeBrachyury sequence will be needed for effective antisense suppression.The introduced antisense sequence in the vector may be at least 15nucleotides in length, and improved antisense suppression typically willbe observed as the length of the antisense sequence increases. Thelength of the antisense sequence in the vector advantageously may begreater than 100 nucleotides, and can be up to about the full length ofthe Brachyury cDNA or gene. For suppression of the Brachyury geneitself, transcription of an antisense construct results in theproduction of RNA molecules that are the reverse complement of mRNAmolecules transcribed from the endogenous Brachyury gene in the cell.

Although the exact mechanism by which antisense RNA molecules interferewith gene expression has not been elucidated, it is believed thatantisense RNA molecules bind to the endogenous mRNA molecules andthereby inhibit translation of the endogenous mRNA. Expression ofBrachyury can also be reduced using small inhibitory RNAs, for instanceusing techniques similar to those described previously (see, e.g.,Tuschl et al., Genes Dev 13, 3191-3197, 1999; Caplen et al., Proc. Nat'lAcad. Sci. U.S.A. 98, 9742-9747, 2001; and Elbashir et al., Nature 411,494-498, 2001). Methods of making siRNA that can be used clinically areknown in the art. Exemplary siRNAs are commercially available fromseveral sources, such as Sigma Aldrich and Dharmacon, and therapeuticsiRNAs can readily be produced using methods known in the art.

Suppression of endogenous Brachyury expression can also be achievedusing ribozymes. Ribozymes are synthetic RNA molecules that possesshighly specific endoribonuclease activity. The production and use ofribozymes are disclosed in U.S. Pat. No. 4,987,071 to Cech and U.S. Pat.No. 5,543,508 to Haselhoff. The inclusion of ribozyme sequences withinantisense RNAs may be used to confer RNA cleaving activity on theantisense RNA, such that endogenous mRNA molecules that bind to theantisense RNA are cleaved, which in turn leads to an enhanced antisenseinhibition of endogenous gene expression.

In certain examples, expression vectors are employed to express theinhibitor nucleic acid, such as the antisense, ribozyme or siRNAmolecule (see above for additional information on vectors and expressionsystems). For example, an expression vector can include a nucleic acidsequence encoding the antisense, ribozyme or siRNA molecule. In aparticular example, the vector contains a sequence(s) encoding bothstrands of a siRNA molecule comprising a duplex. In another example, thevector also contains sequence(s) encoding a single nucleic acid moleculethat is self-complementary and thus forms a siRNA molecule. Non-limitingexamples of such expression vectors are described in Paul et al., NatureBiotechnology 19:505, 2002; Miyagishi and Taira, Nature Biotechnology19:497, 2002; Lee et al., Nature Biotechnology 19:500, 2002; and Novinaet al., Nature Medicine, online publication Jun. 3, 2003, and additionalvectors are described above.

In other examples, inhibitory nucleic acids, such as siRNA moleculesinclude a delivery vehicle, including inter alia liposomes, foradministration to a subject, carriers and diluents and their salts, andcan be present in pharmaceutical compositions. Nucleic acid moleculescan be administered to cells by a variety of methods known to those ofskill in the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other deliveryvehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules,and bioadhesive microspheres, or by proteinaceous vectors (see, forexample, O'Hare and Normand, International PCT Publication No. WO00/53722, see also the additional methods described above).

Alternatively, the nucleic acid/vehicle combination can be locallydelivered such as into a tumor by direct injection or by use of aninfusion pump. Direct injection of the nucleic acid molecules of thedisclosure, whether subcutaneous, intramuscular, or intradermal, cantake place using standard needle and syringe methodologies, or byneedle-free technologies such as those described by Barry et al.,International PCT Publication No. WO 99/31262. Other delivery routesinclude, but are not limited to, oral delivery (such as in tablet orpill form), intrathecal or intraperitoneal delivery (see below). Forexample, intraperitoneal delivery can take place by injecting thetreatment into the peritoneal cavity of the subject in order to directlydeliver the molecules to the tumor site. More detailed descriptions ofnucleic acid delivery and administration are provided in Sullivan etal., PCT WO 94/02595, Draper et al., PCT Publication No. WO93/23569,Beigelman et al., PCT WO99/05094, and Klimuk et al., PCT Publication No.WO 99/04819, all of which are incorporated by reference herein.

Alternatively, certain siRNA molecules can be expressed within cellsfrom eukaryotic promoters. Those skilled in the art will recognize thatany nucleic acid can be expressed in eukaryotic cells using theappropriate DNA/RNA vector (see above). The activity of such nucleicacids can be augmented by their release from the primary transcript byan enzymatic nucleic acid (Draper et al., PCT Publication No. WO93/23569, and Sullivan et al., PCT Publication No. WO 94/02595).

In other examples, siRNA molecules can be expressed from transcriptionunits (see for example, Couture et al., 1996, TIG 12:510) inserted intoDNA or RNA vectors. The recombinant vectors can be DNA plasmids or viralvectors. siRNA expressing viral vectors can be constructed based on, forexample, but not limited to, adeno-associated virus, retrovirus,adenovirus, lentivirus or alphavirus. In another example, pol III basedconstructs are used to express nucleic acid molecules of the invention(see for example, Thompson, U.S. Pat. Nos. 5,902,880 and 6,146,886 andothers described above).

The recombinant vectors capable of expressing the siRNA molecules can bedelivered as described above, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of nucleic acid molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the siRNA molecule interactswith the target mRNA and generates an RNAi response. Delivery of siRNAmolecule expressing vectors can be systemic, such as by intravenous orintramuscular administration, by administration to target cellsexplanted from a subject followed by reintroduction into the subject, orby any other means that would allow for introduction into the desiredtarget cell.

Diagnostic Methods

A method is also provided herein for detecting Brachyury in a biologicalsample. The method includes contacting the sample with one or more of anantibody that specifically binds Brachyury to form an antibody-Brachyurycomplex. The presence or absence of the complex is detected. The methodsare of use to improve the confidence of a tissue diagnosis, such as toconfirm a diagnosis, or to determine the origin of a tumor. Thus, themethod disclosed herein can be used to confirm the diagnosis of a tumorof the small intestine, stomach, kidney, bladder, uterus, ovaries,testes lung, colon and prostate tumors. The methods disclosed herein canbe used to confirm the diagnosis of a B cell tumor, such as chroniclymphocytic leukemia (CLL), Epstein-Barr virus transformed B cells,Burkitt's and Hodgkin's lymphomas. The methods disclosed herein can alsobe used to determine if the origin of a tumor, such as to determine if ametastatic cancer is of small intestine, stomach, kidney, bladder,uterus, ovaries, testes lung, colon, prostate or B cell origin.

In addition, expression of Brachyury is associated with epithelial-tomesenchymal transition. Thus, the methods disclosed herein can be usedto determine the likelihood of tumor cell migration and invasion.

The methods can include selecting a subject in need of diagnosis, suchas a subject with a tumor, and obtaining a sample from this subject. Inseveral examples, the methods include selecting a subject with a tumorof the small intestine, stomach, kidney, bladder, uterus, ovaries,testes lung, colon or prostate, and obtaining a sample from thissubject. In additional examples, the method includes selecting a subjectwith a tumor of B cell origin, such as chronic lymphocytic leukemia(CLL), a B cell lymphoma, Burkitt's lymphoma or a Hodgkin's lymphoma,and obtaining a sample from this subject.

The sample can be any sample, including, but not limited to, tissue frombiopsies, autopsies and pathology specimens. Biological samples alsoinclude sections of tissues, such as frozen sections taken forhistological purposes. Biological samples further include body fluids,such as blood, serum, spinal fluid or urine. A biological sample istypically obtained from a mammal, such as a rat, mouse, cow, dog, guineapig, rabbit, or primate. In one embodiment, the primate is macaque,chimpanzee, or a human. In some embodiments, a histological section isutilized, and an immunohistochemical assay is performed.

Antibodies that specifically bind Brachyury are known in the art.Antibodies include polyclonal and monoclonal antibodies. In someembodiments, an antibody fragment, such as an Fv fragment is utilized.In a further embodiment, the antibody is labeled (such as with afluorescent, radioactive, or an enzymatic label). In additionalexamples, the antibodies can be conjugated to compounds including, butnot limited to, enzymes, magnetic beads, colloidal magnetic beads,haptens, fluorochromes, metal compounds or radioactive compounds.

Methods of determining the presence or absence of a protein are wellknown in the art. Assays of use include, but are not limited to,radioimmunoassays (RIAs), enzyme linked immunosorbant assays (ELISA), orimmunohistochemical assays. The method for detecting Brachyury in abiological sample generally includes the steps of contacting thebiological sample with an antibody which specifically reacts, underimmunologically reactive conditions, to Brachyury. The antibody isallowed to specifically bind under immunologically reactive conditionsto form an immune complex, and the presence of the immune complex (boundantibody) is detected directly or indirectly. A control cell, such as anon-transformed cell or section of the same tissue type, can be includedas a control.

Reagents for the Detection of Cells that Express CD8 (CD8+) Cells thatSpecifically Bind Brachyury

Reagents are provided herein for the detection of CD8 expressing cellsthat specifically bind Brachyury. These reagents are tetrameric MHCClass I/immunogenic Brachyury polypeptide complexes. These tetramericcomplexes include an immunogenic Brachyury polypeptide that includes atmost twelve consecutive amino acids, wherein the isolated polypeptidecomprises the amino acid sequence set forth as WLLPGTSTX₁ (SEQ ID NO:3), wherein X₁ is a leucine (L) or a valine (V). Specific examples ofimmunogenic Brachyury polypeptide that are ten amino acids in length aredisclosed above. The tetrameric complexes disclosed herein do notinclude additional consecutive amino acids of Brachyury (SEQ ID NO: 1),such that the polypeptide does not include the full length Brachyuryamino acid sequence.

Tetrameric MHC Class I/peptide complexes can be synthesized usingmethods well known in the art (Altmann et al., Science 274:94, 1996,which is herein incorporated by reference). In one specific non-limitingexample, purified HLA heavy chain and β2-microglobulin (β2m) can besynthesized by means of a prokaryotic expression system. One specific,non-limiting example of an expression system of use is the pET system(R&D Systems, Minneapolis, Minn.). The heavy chain is modified bydeletion of the trans-membrane and cytosolic tail and COOH-telininaladdition of a sequence containing the biotin protein ligase (Bir-A)enzymatic biotinylation site. Heavy chain, β2m, and peptide are thenrefolded. The refolded product can be isolated by any means known in theart, and then biotinylated by Bir-A. A tetramer is then produced bycontacting the biotinylated product with strepavidin.

In one embodiment, the strepavidin is labeled. Suitable labels include,but are not limited to, enzymes, magnetic beads, colloidal magneticbeads, haptens, fluorochromes, metal compounds, radioactive compounds ordrugs. The enzymes that can be conjugated to strepavidin include, butare not limited to, alkaline phosphatase, peroxidase, urease andβ-galactosidase. The fluorochromes that can be conjugated to thestrepavidin include, but are not limited to, fluorescein isothiocyanate,tetramethylrhodamine isothiocyanate, phycoerythrin, allophycocyanins andTexas Red. For additional fluorochromes that can be conjugated tostrepavidin, see Haugland, R. P., Molecular Probes: Handbook ofFluorescent Probes and Research Chemicals (1992-1994). The metalcompounds that can be conjugated to the strepavidin include, but are notlimited to, ferritin, colloidal gold, and particularly, colloidalsuperparamagnetic beads. The haptens that can be conjugated to thestrepavidin include, but are not limited to, biotin, digoxigenin,oxazalone, and nitrophenol. The radioactive compounds that can beconjugated to strepavidin are known to the art, and include but are notlimited to technetium 99m (⁹⁹Tc), ¹²⁵I and amino acids comprising anyradionuclides, including, but not limited to, ¹⁴C, ³H and ³⁵S.Generally, strepavidin labeled with a fluorochrome is utilized in themethods disclosed herein.

In one embodiment, suspension of cells including T cells thatspecifically recognize Brachyury is produced, and the cells are reactedwith the tetramer in suspension. In one embodiment, these reagents areused to label cells, which are then analyzed by fluorescence activatedcell sorting (FACS). A machine for FACS employs a plurality of colorchannels, low angle and obtuse light-scattering detection channels, andimpedance channels, among other more sophisticated levels of detection,to separate or sort cells. Any FACS technique can be employed as long asit is not detrimental to the detection of the desired cells. (Forexemplary methods of FACS see U.S. Pat. No. 5,061,620.)

The disclosure is illustrated by the following non-limiting Examples.

Examples Example 1 Materials and Methods

Computer-Based Differential Display (CDD) Analysis.

Comparison of all EST clusters on the human Unigene Built 171 (see theNCBI Unigene website, available on the internet) was conducted by usingthe HSAnalyst program as previously described (Baronova et al., FEBSLett 508(1):143-8, 2001). Unigene EST cluster Hs.389457 corresponds toaccession number NM_(—)003181.

Source of cDNA.

Expression in normal tissues was studied by using Multiple Tissue cDNA(MTC) panels containing sets of normalized cDNAs from pooled normaltissues from several individuals (Clontech, Mountain View, Calif.). Thefollowing panels were used: human MTC Panel I, Panel II, and BloodFractions Panel. Commercially available tumor tissue-derived cDNAs,prepared from different individuals with different tumor types, wereobtained from BioChain Institute Inc. (Hayward, Calif.). Total RNA fromhuman cancer cell lines and normal CD19+ isolated B cells were preparedby using the RNAeasy extraction kit (Qiagen Inc., Valencia, Calif.).

PCR Analysis.

PCR amplification of cDNA panels was carried out with the followingprimers specific for NM_(—)003181:

(SEQ ID NO: 4) E7F 5′-GGGTGGCTTCTTCCTGGAAC-3′ and (SEQ ID NO: 5)E7R 5′-TTGGAGAATTGTTCCGATGAG-3′.

G3PDH specific primers were:

(SEQ ID NO: 6) forward 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′, (SEQ ID NO: 7)reverse 5′-CATGTGGGCCATGAGGTCCACCAC-3′.

The following conditions were used: 1 minute at 95° C., 35 cyclesconsisting of 30 sec at 95° C., 30 sec at 58°, and 1 minute at 72° C.,and 5 minutes elongation at 72° C. The expected size for the Brachyuryand G3PDH products was 172 and 983 bp, respectively. Total RNA derivedfrom human cancer cell lines and normal CD19+ isolated B cells wereamplified by using the TITANIUM One-Step RT-PCR kit (Clontech),following the manufacturer's instructions. Primer sequences were asfollow:

Brachyury, E3F 5′-ACTGGATGAAGGCTCCCGTCTCCTT-3′ (SEQ ID NO: 8), and E8R5′-CCAAGGCTGGACCAATTGTCATGGG-3′ (SEQ ID NO: 9) (Edwards et al., GenomeRes 6(3):226-33, 1996); and

β-actin, forward 5′-ATCTGGCACCACACCTTCTACAATGAG-3′ (SEQ ID NO: 10), andreverse 5′-CGTGGTGGTGAAGCTGTAGCCGCGCTC-3′ (SEQ ID NO: 11). The expectedsize of the PCR products was 568 bp and 356 bp, respectively.

RT-PCR Amplification from NCI-H460 Cells:

Total RNA was prepared from stably transfected NCI-H460 cells containinga control shRNA plasmid or a Brachyury-specific shRNA construct (Br.shRNA clones 1 and 2) by using the RNeasy extraction kit (Qiagen Inc.,Valencia, Calif.), following the manufacturer's recommendations. Five ngof total RNA were amplified by using the TITANIUM One-Step RT-PCR kit(Clontech, Mountain View, Calif.), following the manufacturer'sinstructions. Primer sequences were as follows: Brachyury, E3F5′-ACTGGATGAAGGCTCCCGTCTCCTT-3′ (SEQ ID NO: 8), and E8R5′-CCAAGGCTGGACCAATTGTCATGGG-3′ (SEQ ID NO: 9); and β-actin, forward5′-ATCTGGCACCACACCTTCTACAATGAG-3′ (SEQ ID NO: 10), and reverse5′-CGTGGTGGTGAAGCTGTAGCCGCGCTC-3′ (SEQ ID NO: 11). The expected size ofthe PCR products was 568 and 356 bp, respectively.

Cell cultures.

The human carcinoma cell lines were maintained free of Mycoplasma inRPMI 1640 medium (Invitrogen, Carlsbad, Calif.) supplemented with 10%fetal bovine serum, 2 mM glutamine, and 1× solution ofantibiotic/antimycotic (Invitrogen). Additional cell lines used in thisstudy were the C1R-A2 cell line, which is a human B-cell lymphoblastoidline transfected to express surface HLA-A2 antigen (Shimojo et al., JImmunol 143(9):2939-47, 1989), and the T2 (HLA-A2+) transport-deletionmutant cell line (Salter et al., Embo J 5(5):943-9, 1986).

Peptides.

The computer algorithm from the Bioinformatics and Molecular AnalysisSection of NIH (BIMAS) developed by Parker et al. was used (Parker etal., J Immunol 152(1):163-75, 1994, incorporated by reference herein). Apanel of 9-mer and 10-mer peptides (see Table 2, below) was synthesizedat >90% purity (Biosynthesis, Lewisville, Tex.). The CEA peptide CAP1-6D(YLSGADLNL, SEQ ID NO: 12), the HIV peptide (ILKEPVHGV, SEQ ID NO: 13),and a CEA peptide specific for HLA-A3 were used as controls.

HLA A2 Binding Assay.

Binding of Brachyury-specific peptides T-p1, T-p2, T-p3, and T-p4 (SEQID NOs: 1-4) to HLA-A0201 molecules was evaluated by flow cytometryanalysis of HLA-A02 surface expression on T2 cells. T2 cells (1×10⁶) inserum-free Iscove's modified Dulbecco's medium were incubated in thepresence of various concentrations of each peptide, in 24-well cultureplates at 37° C. with 5% CO₂. After 18 hours in culture, T2 cells wereharvested, washed with 1× phosphate buffered saline (PBS) (Invitrogen)and stained with 20 μl of a FITC-conjugated anti-HLA-A02-specificmonoclonal antibody (MAb) (One Lambda, Inc., Canoga Park, Calif.). AFITC-conjugated IgG2a MAb (BD Biosciences, San Jose, Calif.) was used asan isotype control. Data acquisition and analysis were conducted on aFACSCalibur™ system using the CELLQuest™ software (BD Biosciences).Results were expressed as mean fluorescence intensity (MFI) collected ona log scale. To measure the half-life of major histocompatabilitycomplex (MHC)-peptide complexes, T2 cells were incubated for 18 hours inthe presence of 25 μM of each peptide, subsequently washed free ofunbound peptides and incubated for various time points in presence of 10μg/ml of Brefeldin A. Flow cytometry was conducted as described above.Assuming first order kinetics, the log₂ of MFI/MFI₀ (MFI is thefluorescence at each time point and MFI₀ the initial fluorescence attime 0) was plotted against time (minutes). The decay rate constant wascalculated as the slope of the linear regression for each curve and thehalf-life of each peptide-MHC complex was calculated as the inverse ofthe ratio 1/decay rate constant.

Culture of DCs from Peripheral Blood Mononuclear Cells (PBMCs).

Peripheral blood used in this study was collected from healthy donorsand cancer patients. Peripheral blood mononuclear cells (PBMCs) wereisolated from leukapheresis samples by centrifugation on a Ficolldensity gradient (LSM Lymphocyte Separation Medium, ICN BiochemicalsInc., Aurora, Ohio). For the preparation of dendritic cells (DCs), PBMCswere resuspended in AIM-V medium (Invitrogen) and allowed to adhere tothe surface of T-150 flasks (Corning Costar Corp., Cambridge, Mass.).After 2 hours at 37° C., the non-adherent cell fractions were removedand the adherent cells were cultured in AIM-V medium containing 100ng/ml of recombinant human GM-CSF (rhGM-CSF) and 20 ng/ml of recombinanthuman IL-4 (rhIL-4) for 7 days.

Generation of T-Cell Lines.

To generate Brachyury-specific cytotoxic T cells (CTLs), peptide-pulsedirradiated (30 Gy) DCs were used as antigen presenting cells (APCs) withautologous, non-adherent cells used as effector cells at aneffector-to-APC ratio of 10:1. Cultures were maintained for threeinitial days in medium containing 10% human AB serum, and fouradditional days in the same medium supplemented with 20 U/ml ofrecombinant human IL-2. After a seven-day culture period, designated asan in vitro stimulation (IVS) cycle, cells were re-stimulated asdescribed above.

Detection of Cytokines.

After three IVS cycles, CD8+ T cells that were negatively isolated byusing a CD8+ isolation kit (Miltenyi Biotec, Auburn, Calif.) werestimulated for 24 hours in the presence of peptide-pulsed autologousDCs. Culture supernatants were analyzed for the presence of IFN-γ byusing an enzyme linked immunosorbant assay (ELISA) kit (BiosourceInternational Inc., Camarillo, Calif.). Results were expressed in pg/ml.

Cytotoxic Assay.

Target cells were labeled with 50 μCi of ¹¹¹Indium-labeled oxyquinoline(Amersham Health, Silver Spring, Md.) for 15 minutes at roomtemperature. Target cells in medium containing 10% human AB serum wereplated at 3×10³ cells per well, in 96-well rounded-bottom cultureplates. Labeled C1R-A2 or T2 cells were incubated with peptides at theindicated concentrations for 60 minutes at 37° C. in 5% CO₂ before theaddition of effector cells. No peptide was added when carcinoma cells orCD19+ B cells were used as targets. CD8+ T cells negatively isolatedfrom T-cell cultures were used as effector cells, at variouseffector-to-target (E:T) cell ratios. When target cells were C1R-A2 orT2, co-cultures were incubated at 37° C. in a 5% CO2 atmosphere for 6hours as previously described (Tsang et al., Clin Cancer Res11(4):1597-607, 2005); when carcinoma cell lines were used as targets,co-cultures were incubated as previously described (Tsang et al., supra)in the same conditions for a period of 16 hours. Cytotoxic assaysemploying normal donor CD19+ B cells as targets were conducted for 5hours as previously described (Palena et al., Blood 106(10):3515-23,2005), due to the high levels of spontaneous release observed after a16-hour incubation period. Supernatants were harvested and the ¹¹¹Inreleased was measured by gamma counting. Spontaneous release wasdetermined by incubating the target cells with medium alone, andcomplete lysis by incubating the target cells with 2.5% Triton X-100.All determinations were performed in triplicate, and standard deviationswere calculated. Specific lysis was calculated as follows: specificlysis (%)=[(observed release−spontaneous release)/(completerelease−spontaneous release)]×100.

Generation of Stably Transfected NCI-H460 Cells:

The NCI-H460 cell line, originally derived from a patient with a largecell carcinoma of the lung, was obtained from the American Type CultureCollection (ATCC, Manassas, Va.) and maintained in RPMI-1640 mediumsupplemented with 1× Antibiotic/Antimycotic solution (Invitrogen,Carlsbad, Calif.) and 10% fetal bovine serum (FBS, Gemini Bio-ProductsWest Sacramento, Calif.). Cells (1×10⁶) were transfected using thenucleofector device and technology (Amaxa Biosystems, Gaithersburg, Md.)with 1 μg of purified, linearized DNA plasmid encoding for anon-targeting shRNA (designated as control shRNA) or twoBrachyury-specific targeting shRNA constructs (designated as Br. shRNAclones 1 and 2), following the recommendations of the manufacturers.After 48 hours in culture, stably transfected cells were selected inRPMI-1640 medium containing 10% FBS and 1 μg/ml of puromycin (SigmaAldrich, St. Louis, Mo.).

Western Blot Analysis of Mesenchymal and Epithelial Markers:

Protein extracts were prepared from NCI-H460 cells stably transfectedwith the control shRNA and Brachyury-specific shRNA (clone 2) by usingthe Ripa Lysis Buffer kit (Santa Cruz Biotech, Santa Cruz, Calif.)following the manufacturer's instructions. Protein concentration wasdetermined by using the BCA Protein Assay kit (Thermo Scientifics,Rockford, Ill.).

Ten micrograms (mg) of proteins from each sample were resolved on 4-12%polyacrylamide gradient pre-cast gels and subsequently transferred tonitrocellulose membranes (Invitrogen). Blots were blocked with freshlymade 0.5% casein in PBS for 1 hour at room temperature. Subsequently,blots were probed with 1:500-1:1000 dilution of primary antibodies in0.5% casein solution overnight at 4° C. Antibodies were anti-humanfibronectin, vimentin, g-catenin, and b-actin (BD-Biosciences, San Jose,Calif.). Blots were washed 3 times with PBS and incubated with a 1:5000dilution of horseradish peroxidase (HRP)-conjugated secondary anti-mouseIgG antibody (Invitrogen) for 1 hour at room temperature. Blots werewashed 5 times with PBS/Tween 20 and were developed using the WesternLighting chemiluminescent detection reagent (PerkinElmer, Boston Mass.)and autoradiographs were obtained.

Migration and Invasion Assays:

The migratory abilities of NCI-H460 cells stably transfected with acontrol shRNA or a Brachyury-specific shRNA (Br.shRNA clone 2) wereexamined in vitro using Blind Well Chambers (Neuroprobe, Gaithersburg,Md.) with 12 micrometer-pore size polycarbonate filters. Briefly,RPMI-1640 medium containing 10% fetal bovine serum (FBS) was added tothe lower chambers, and cells (1×10⁵ cells, 300 μl in RPMI medium freeof serum) to the upper chambers. For the invasion assays, polycarbonatefilters were pre-coated with a 1:1 dilution of Matrigel (BD Biosciences)and serum free RPMI-1640 medium. Experiments were conducted intriplicate samples of each cell line. After incubation for 48 hours at37° C., the upper side of the filters was extensively cleaned withcotton tips, filters were removed from the chambers, fixed, and stainwith Diff-Quik stain (Dade Behring Inc., Newark, Del.). The number ofcells associated with the lower side of the membranes was evaluated bydirect counting of five random 100× objective fields. Each barrepresents the results for each replicate assay±SEM.

Example 2 Computer-Based Prediction

In silico profiling of gene expression in the human Unigene Built 171was conducted as previously described (Baranova et al., FEBS Lett508(1):143-8, 2001) by using the HSANALYST™ software tool. An algorithmexecuted by the program returned a list of candidate EST clusters thatcontained >10 ESTs with >90% of the ESTs derived from tumor libraries.Among them, the cluster Hs.389457 contained the whole mRNA sequenceencoding for the human Brachyury gene (mouse Brachyury homolog). From atotal of 55 ESTs included in this cluster, 50 ESTs corresponded totumor-derived libraries constructed from lung carcinoma cell lines,germ-cell tumors, chronic lymphocytic leukemia B cells, and breastcancer. Two normal tissue-derived ESTs found in the cluster Hs.389457belonged to a library constructed from pooled RNA from fetal lung,testis, and normal B cells. The other three ESTs in the cluster weredesignated as “undefined,” since they lacked tissue origin descriptions.

Example 3 Confirmation of Expression

The computer-based predictions of the expression of Brachyury mRNA werethen verified by RT-PCR analysis of Brachyury expression in a range ofnormal and malignant human tissues. Most normal tissue-derived cDNAsamples, as predicted by the algorithm, showed no Brachyury mRNAexpression (FIGS. 1A and 1B). Very weak signals, however, were observedwith cDNA derived from normal testis, spleen (FIG. 1A), and restingCD19+ purified cells (FIG. 1B). These results were also in accordancewith the software's prediction that two out of 55 ESTs in the clusterbelonged to a library prepared from pooled testis, fetal lung, andnormal B lymphocytes.

The expression of Brachyury in normal B cells was further evaluated inCD19+ samples isolated from various healthy donors; weak amplificationwas observed in four out of nine samples analyzed when using 1 microgramof total RNA and 35 cycles of PCR amplification.

In contrast, RT-PCR amplification of cDNA samples derived fromtumor-tissues demonstrated relatively high levels of Brachyury mRNAexpression in carcinomas of the esophagus, stomach, small intestine,kidney, bladder, uterus, ovary, and testis (FIG. 1C), and a weak signalin a lung carcinoma-derived sample. PCR products derived from two of thereactions were subsequently sequenced to confirm the gene and rule oatthe possibility of non-specific amplification. The expression ofBrachyury was further analyzed in total RNA derived from 30 humancarcinoma cell lines (Table 1).

TABLE 1 RT-PCR expression of human Brachyury in human tumor cell linesBrachyury Tumor type Tumor cell line mRNA¹ Lung H441 ++ NCI-H460 ++H226 + NCI-H520 + SW900 − Colon SW480 ++ SW620 ++ Colo 201 + Colo 205 +CaCo2 + SW403 + T-84 + SW948 − SW1463 − HT-29 − SW1116 − ProstateLNCAP + PC-3 + DU145 + Pancreatic Capan-2 + Paca-2 − BxPC3 − PANC-1 −ASPC-1 − Breast MCF-7 − MA-MB-231 − Ovarian SW626 ++^(a) NIH-OVCAR3 −SK-OV3 − Osteosarcoma U2OS − ¹Expression of Brachyury mRNA is shownrelative to the expression of β-actin as being negative (−), positive(+), or strongly positive (++). ^(a)There is conflicting evidence thatthis cell line may be of colonic origin. See Furlong et al., J NatlCancer Inst 91(15): 1327-8, 1999.

Brachyury mRNA expression was observed in most of the lungcancer-derived, colon cancer-derived, and prostate cancer-derived tumorcell lines (Table 1). These results thus validated the CDD predictionsthrough RT-PCR and confirmed expression of Brachyury in several tumorsbut not in normal tissues.

Reverse transcriptase polymerase chain reaction (RT-PCR) analysisdemonstrated Brachyury expression in tumors of the small intestine,stomach, kidney, bladder, uterus, ovary, and testis, as well as in celllines derived from lung, colon, and prostate carcinomas, but not in thevast majority of the normal tissues tested. Elevated Brachyury mRNAexpression was also detected in most of the B-cell malignanciesexamined, including chronic lymphocytic leukemia (CLL) cells,Epstein-Barr virus (EBV)-transformed B-cell lines, Burkitt's andHodgkin's lymphoma cell lines. Quantitative real-time PCR analysisshowed elevated expression of Brachyury mRNA in CD19⁺ cells isolatedfrom 13/25 CLL patients, as compared with very low, if any, level ofexpression in B lymphocytes isolated from peripheral blood from healthydonors or a panel of normal human tissues. A time-course infection ofnormal B-lymphocytes with EBV showed that Brachyury mRNA expression isinduced as early as 48 hours post-infection and is maintained inlong-term cultures of transformed B-cell lines. EBV, a lymphotropichuman herpesvirus, is linked to various clinical disorders includinghuman neoplasms of hematological origin such as lymphomas inimmunocompromised individuals and posttransplant lymphoproliferativedisorders, and those of epithelial origin such as nasopharyngealcarcinomas and gastric adenocarcinomas. These results suggest thatBrachyury is a potential tumor target for hematological malignancies ofB-cell origin and, in particular, for EBV-associated malignancies. Theresults also demonstrate that elevated Brachyury expression (as comparedto a control cell of the same tissue type) serves as a marker forconfirmation of diagnosis of tumors of these types. In addition toimproving the confidence with which a tissue diagnosis of the cancers ismade, the detection of Brachyury over-expression in a metastatic lesionhelps identify potential sites of primary tumor so that furtherdiagnostic tests of these potential sites can be more cost effectivelyconducted and therapy (such as surgical excision of the primary tumor)more quickly achieved. Hence detection of Brachyury can be used inmethods of diagnosing and treatment of cancers characterized byBrachyury expression.

Example 4 Production of Brachyury Immunogenic Peptides that Bind MHC

The amino acid sequence of the Brachyury protein was then analyzed forHLA-A0201 peptide-binding prediction by using a computer algorithm fromBIMAS. The top-ranking candidate peptides generated by the program,including three 9-mers and a 10-mer whose amino acid sequences andalgorithm scores are presented in Table 2, were selected for furtherstudies.

TABLE 2  HLA-A0201 peptide motif search using BIMAS software StartPeptide Residues position* Sequence Score† T-p1  9-mer 345 SQYPSLWSV389.26 (SEQ ID NO: 14) T-p2  9-mer 246 WLLPGTSTL 363.59 (SEQ ID NO: 15)T-p3  9-mer 422 RLIASWTPV 118.24 (SEQ ID NO: 16) T-p4 10-mer  86AMYSFLLDFV 996.36 (SEQ ID NO: 17) *Start position corresponds to theamino acid position in the protein sequence. †Estimate of half-timedisassociation of a molecule containing this subsequence.

In silico-predicted epitopes were then assessed for binding to the MHCmolecules in a cell-based assay. TAP-deficient T2 (HLA-A2+) cells wereincubated in the presence of 25 μM of each peptide and subsequentlytested for cell surface MHC-stabilization by bound peptides. Flowcytometry staining of HLA-A02 (FIG. 2A) demonstrated that all fourcandidate peptides predicted by the algorithm efficiently bound toHLA-A02 molecules when compared to positive and negative controlpeptides. Peptides with the highest binding to T2 cells (T-p2, T-p3, andT-p4) were selected for further studies. The half-life of eachpeptide-MHC complex was determined; T2 cells were incubated overnight inthe presence of 25 μM of each peptide followed by the addition ofbrefeldin A and subsequent evaluation of cell surface staining ofHLA-A02 at various time points. MHC-peptide complexes involving Tp-2have a half-life of 514 minutes, similar to that of the positive controlpeptide (CAP1-6D). In contrast, MHC-peptide complexes involving T-p3 andT-p4 showed shorter half-lives of 225 and 312 minutes, respectively(FIG. 2B).

Example 5 Immunogenicity

Once the ability of the predicted peptides to bind HLA-A02 molecules wasdemonstrated, the immunogenicity of peptides T-p2, T-p3, and T-p4 wasinvestigated by evaluating their ability to induce specific CTLs invitro. Irradiated DCs pulsed with 25 μM of each peptide were used tostimulate autologous T cells from a healthy donor's PBMCs. After threein vitro stimulations (IVS), isolated CD8+ T cells were subsequentlystimulated for 24 hours in the presence of autologous DCs alone or DCspulsed with each of the “inducer” peptides (T-p2, T-p3, or T-p4) or anirrelevant HIV-peptide. Of the three peptides tested, T-p2 and T-p3induced antigen-specific CTLs able to release IFN-γ upon stimulationwith the specific peptide (FIG. 3A). Both CTL lines were then tested fortheir cytotoxic activity against peptide-pulsed HLA-A0201+ targets. Asshown in FIG. 3B, only T cells generated with the T-p2 peptide were ableto specifically lyse peptide-pulsed target cells, consistent with thepeptide's ability to form stable MHC complexes compared with T-p3 andT-p4. Titration of the cytotoxic activity of the T-p2 CTLs showedcytotoxic responses at peptide concentrations as low as 1 nM (FIG. 3C).Cytotoxic lysis of normal B lymphocytes was also analyzed since lowexpression of Brachyury was detectable in CD19+ cells isolated fromvarious healthy donors. No lysis was observed with any of the normal Bcells analyzed from five different healthy donors.

The cytolytic activity of the T-p2-specific CTLs was then tested againstseveral tumor targets. Tumor cell lines used as targets included thelung carcinoma cells H441 (HLA-A0201+/T antigen+) and NCI-H460 (HLA-A24,68+/T antigen+), the colorectal carcinoma line SW1463 (HLA-A0201+/Tantigen−), and the pancreatic carcinoma cells AsPC-1 (HLA-A02−). CTLsderived with the T-p2 epitope were highly efficient at killing H441tumor cells, while no lysis was observed against the other cell lines.MHC-restriction was shown by the observation that the H460 tumor cellline that is highly positive for Brachyury but HLA-A0201 negative wasnot killed by the Tp-2 CTLs (FIG. 4A). Conversely, the tumor cell lineSW 1463 served as an antigen-specific control, since it is negative forthe expression of Brachyury but positive for the expression ofHLA-A0201. Similarly, the control AsPC-1 (HLA-A0201−) cells were alsonot killed by the Brachyury-specific T cells. These results indicatethat T cells that have been expanded in vitro in the presence of theT-p2 peptide are able to specifically lyse those tumor cells thatexpress Brachyury within the correct MHC-class I context. As shown inFIG. 4B, T-p2 CTLs-mediated killing of H441 tumor cells was blocked byantibodies directed against the MHC-class I molecules but not theMHC-class II molecules, further confirming the MHC-class I restrictionof the observed lysis.

The Tp-2 peptide was then tested for in vitro expansion ofBrachyury-specific T cells from PBMC of four additional healthy donors.Tp-2-specific CTLs were induced from two out of five healthy donorstested.

Generation of T-p2 specific CTLs was also successfully carried out fromPBMCs of two cancer patients. T cells isolated from PBMCs of acolorectal cancer patient (designated as patient 1) and an ovariancancer patient (designated as patient 2) were stimulated in vitro forthree cycles in the presence of autologous, irradiated T-p2-pulsed DCsas described in Example 1. CD8+ T cells negatively isolated from thesecultures were assayed for cytotoxic activity against tumor cells. Asshown in FIGS. 4C and 4D, after three IVS both CTL lines were able tolyse H441 tumor cells. After five IVS, CTLs derived from both patientswere tested for their ability to lyse additional tumor cell linespositive for the expression of Brachyury. As shown in FIG. 4E,T-p2-specific CTLs derived from patient 1 were able to lyse LNCAP cells(HLA-A2+/Brachyury+) in an HLA-A02 restricted way, as denoted by theblocking of cytotoxic killing in presence of anti-HLA-A02 but not inpresence of a control IgG. FIG. 4F shows that T-p2 cells expanded fromthe blood of patient 2 were able to lyse H441, SW620, and SW480 tumorcells, all of them being Brachyury+ and HLA-A2+. On the other hand,lysis of SW403 cells, which are HLA-A2+ and express lower levels ofBrachyury mRNA (FIG. 4E), was only minimal. Altogether, Tp-2 cellsderived from healthy individuals and cancer patients were able to lyse4/5 Brachyury positive tumors, while no lysis was observed for controltumor cells that were (a) HLA-A2−/Brachyury+ (NCI-H460), or (b)HLA-A2+/Brachyury-(SW1463).

In conclusion, the results demonstrated that T-p2-specific T cellsgenerated from both healthy donors and cancer patients were able torecognize and mediate cytotoxic lysis of tumor cells that endogenouslyexpress the Brachyury protein.

As demonstrated herein, high-throughput gene expression analysis intumors versus normal tissues constitutes a relatively new approach forthe identification of therapeutic cancer targets. Computer programs havebeen emerging for mining of EST databases that use publicly availableinformation from the vast collection of ESTs (Scheurle et al., CancerRes 2000, 60(15):4037-4043). As the frequency of ESTs in a cDNA libraryappears to be proportional to the abundance of associated transcripts inthe tissue from which the library was prepared (Audic and Claverie,Genome Res 1997; 7:986-995), data on ESTs expression can be correlatedwith tissue-related or disease-related gene expression signatures. Inthe present studies, data mining software tool (HSANALYST™) wassuccessfully used for the identification of Unigene EST clusterHs.389457, corresponding to the human gene Brachyury, as a tumorantigen, and validated the in silico prediction by RT-PCR in a set ofnormal and tumor tissues and cancer cell lines. Expression of Brachyurywas shown to be elevated in tumors of the small intestine, stomach,kidney, bladder, uterus, ovary, and testis, and in the majority of celllines derived from lung, colon, and prostate carcinomas. Because of thehigh grade of conservation among members of the T-box family, BLASTanalysis of the primers sequence was conducted to discard any possibleamplification of sequences derived from other members of the T-boxfamily, and the fidelity of the amplified band was confirmed by DNAsequencing. The high levels of expression of Brachyury in tumorscontrasted with its lack of expression in most normal adult tissues,with the exception of low levels observed in testis, spleen, and CD19+(resting) lymphocytes. Without being bound by theory, the weak signal inspleen could be attributable to the presence of CD19+ cells.

The affinity prediction method from BIMAS was applied in the studiespresented herein for identifying Brachyury peptides with high affinitybinding for HLA-A0201. All four top-ranked peptides effectively bound toHLA-A0201 molecules, although peptide-MHC complexes showed differencesin their decay rate. Tp-2 was the only peptide, however, able to expandCTLs in vitro that are capable of releasing IFN-γ in response topeptide-specific stimulation and lysing peptide-pulsed targets with highefficiency. This peptide also showed the maximum stability of binding toHLA-A0201, which could result in increased immunogenicity.

The lung carcinoma cell line H441 was effectively lysed in the presenceof Brachyury-specific CTLs even at a low ratio of effector Tcells-to-targets, in an antigen-specific and MHC-restricted manner.Furthermore, it was demonstrated that Brachyury-T-p2-specific CTLs canbe expanded in vitro from PBMCs of a colorectal cancer patient and anovarian carcinoma patient, demonstrating that Brachyury is of use as atherapeutic target for cancer vaccine regimens. Thus, it has beendemonstrated that (a) a T-box transcription factor and (b) a moleculeimplicated in mesodermal development, (such as epithelial-to mesodermaltransition, EMT), can be a potential target for human T-cell mediatedcancer immunotherapy.

Example 6 Stable Knockdown of Brachyury Expression Induces aMesenchymal-to-Epithelial Transition in NCI-H460 Lung Carcinoma CancerCells

In order to evaluate whether Brachyury plays a role on modulating theepithelial-to-mesenchymal (EMT) program, Brachyury expression was stablysilenced in NCI-H460 lung carcinoma cells that normally express highlevels of Brachyury mRNA. RT-PCR analysis confirmed the silencing ofBrachyury expression in cells transfected with the Brachyury-specificshRNA clones 1 and 2, as compared with cells transfected with thenon-targeting shRNA control construct (FIG. 5A), with the clone 2showing a higher level of silencing of Brachyury expression.

Western blot analysis of expression of various epithelial andmesenchymal markers showed that silencing of Brachyury expressionresulted in marked decreases on the expression of fibronectin andvimentin, both markers of a mesenchymal phenotype. The expression of theepithelial marker γ-catenin, on the other hand, was enhanced as a resultof Brachyury's silencing. Therefore, at the biochemical level, silencingof Brachyury expression in NCI-H460 cells repressed expression ofmesenchymal markers and concomitantly elicited expression of epithelialmarkers, changes typically observed during a mesenchymal-to-epithelial(MET) transition.

Example 7 Loss of Brachyury Impairs the Migratory and InvasiveProperties of NCI-H460 Lung Carcinoma Cells In Vitro

Boyden-chamber transwell assays were also performed in order todetermine whether Brachyury expression in NCI-H460 cells modulates themigratory and invasive characteristics of these cells. As shown in FIG.6, NCI-H460 cells stably silenced for Brachyury expression (Br. shRNAclone 2) showed a significant reduction on their migratory ability (FIG.6A) as well as a marked reduction on their ability to degrade and invadethe extracellular matrix (FIG. 6A). These results support the conclusionthat the T-box transcription factor Brachyury can serve as a modulatorof the mesenchymal phenotype of tumor cells and could programmetastasis-associated cellular qualities.

Example 8 Expression of Brachyury in Hematological Malignancies

The expression of Brachyury in chronic lymphocytic leukemia (CLL) aswell as other hematological malignancies (Hodgkin's lymphoma,non-Hodgkin's lymphoma, Burkitt's lymphoma) was investigated. CD19+ cellwere isolated from CLL patients. The relative expression of Brachyury inCD19+ B cells was evaluated by quantitative real-time polymerase chainreaction (PCR), using primers to human Brachyury and GAPDH. Theexpression of Brachyury in CD19+ cells isolated from CLL patients was0.318+/−0.752, while the expression of Brachyury in healthy donors was0.019+/−0.023. It was also demonstrated that Brachyury was expressed onCD5+CD19+ leukemia cells. Using reverse-transcriptase PCR (RT-PCR)Brachyury was detected in EBV transformed B Cell lines, (B-EBV 701,B-EBV 1383, C1R) as well as in Burkitt's lymphoma (DAUDI and RAJI) andHodgkin's lymphoma (RPMI 6666) cell lines.

B lymphocytes from healthy donors were infected in vitro with EBVsupernatants. The RNA was isolated over time and analyzed for Brachyuryexpression (by RT-PCR). Brachyury was expressed at 72 and 96 hoursfollowing EBV infection. In the tree subject tested, Brachyuryexpression also could be detected six days after EBV infection.

Thus, elevated expression of Brachyury mRNA was detected in CD19+ cellsfrom CLL patients as compared to CD19+ cells from healthy donors. 13/25CLL patients showed increased levels of Brachyury as compared to normalB cells. No correlation was found between the level of Brachyuryexpression and the white blood cell count or Rai stage. Expression ofBrachyury was also elevated in LCL (EBV-transformed B cell) lines,Burkitt's lymphoma cell lines and the Hodgkin's lymphoma line RPMI16666.EBV infection of normal B lymphocytes resulted in expression ofBrachyury mRNA. Brachyury expression could be detected as early as 48hours after infection; Brachyury expression peaked at 72 hourspost-infection.

Example 9 Inhibition of Tumor Growth or Metastasis Using a SpecificBinding Agent

This example describes methods that can be used to significantly reducetumor growth or metastasis in a subject with a tumor of epithelialorigin, such as small intestine, kidney, bladder, uterus, ovary ortestis, or in carcinomas, such as lung, colon and prostate carcinomas,or in a tumor of B-cell origin, such as chronic lymphocytic leukemia,Burkitt's lymphoma or Hodgkin's lymphoma.

Based upon the teaching disclosed herein, tumor growth or metastasis canbe reduced or inhibited by administering a therapeutically effectiveamount of a composition, wherein the composition comprises a specificbinding agent for Brachyury, thereby reducing or inhibiting tumor growthor metastasis in the subject.

In an example, a subject who has been diagnosed with the tumor isidentified and selected for treatment. Following subject selection, atherapeutic effective dose of the composition including the specificbinding agent is administered to the subject. For example, a therapeuticeffective dose of a specific binding agent for Brachyury is administeredto the subject to inhibit tumor growth and/or metastasis. In an example,the specific binding agent is a siRNA. In a further example, thespecific binding agent is an antisense molecule. The amount of thecomposition administered to prevent, reduce, inhibit, and/or treat thetumor depends on the subject being treated, the severity of thedisorder, and the manner of administration of the therapeuticcomposition. Ideally, a therapeutically effective amount of an agent isthe amount sufficient to prevent, reduce, and/or inhibit, and/or treatthe tumor in the subject without causing a substantial cytotoxic effectin the subject.

In one specific example, siRNAs are administered at according to theteachings of Soutschek et al. (Nature Vol. 432: 173-178, 2004) orKarpilow et al. (Pharma Genomics 32-40, 2004) both of which are hereinincorporated by reference in their entireties. In other examples, thesubject is administered the therapeutic composition daily for a periodof at least 30 days, such as at least 2 months, at least 4 months, atleast 6 months, at least 12 months, at least 24 months, or at least 36months.

Subjects will monitored by methods known to those skilled in the art todetermine tumor responsiveness to the siRNA or antisense. It iscontemplated that additional agents can be administered, such asantineoplastic agents in combination.

Example 10 Retrospective Study

In order to determine the correlation of Brachyury expression with theprognosis of a tumor, samples of lung, colon, breast, and prostatecancer tissues, among others, are analyzed by real-time PCR to determinethe expression of Brachyury mRNA, as described above. Commerciallyavailable cDNA panels (TissueScan Real-Time Disease qPCR Arrays, OrigeneTechnologies, Rockville, Md.) containing cDNA prepared from individualtumor samples are tested with Brachyury- and GAPDH-specific PCR primersto obtain a quantitative expression of Brachyury mRNA in each sample.Each cDNA panel includes information on tumor grade and stage (Samplesfrom stages 1A-IV for lung cancer; stages I to IV for prostate cancer;stages I to IV for colon cancer; and stages 0 to IV for breast cancerwill be tested, for example). Brachyury mRNA is preferentially expressedin samples of tumors from patients diagnosed to be at higher stages thanin samples of tumors from patients diagnosed to be at lower stages.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described invention. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

1.-21. (canceled)
 22. An antisense molecule or an siRNA thatspecifically binds a nucleotide sequence encoding SEQ ID NO: 1 for usein a method of inhibiting growth or metastasis of a tumor in a subject,comprising selecting a subject having the tumor; and administering atherapeutically effective amount of the antisense molecule or siRNA tothe subject having the tumor, thereby inhibiting growth or metastasis ofthe tumor in the subject, wherein the cancer cell is from a cancer ofthe small intestine, stomach cancer, kidney cancer, bladder cancer,uterine cancer, ovarian cancer, testicular cancer, lung cancer, breastcancer, bronchial tube cancer, colon cancer, prostate cancer, or a Bcell tumor.
 23. The antisense molecule of claim 22, wherein the cancercell is from a B cell tumour, wherein the B cell tumor is chroniclymphocytic leukemia.
 24. The antisense molecule or siRNA of claim 22,comprising administering to the subject a therapeutically effectiveamount of an siRNA.
 25. The antisense molecule or siRNA of claim 22,further comprising administering to the subject a therapeuticallyeffective amount of an isolated polypeptide comprising at most twelveconsecutive amino acids, wherein the isolated polypeptide comprises theamino acid sequence set forth as one of: (a) WLLPGTSTX₁ (SEQ ID NO: 3),wherein X₁ is a leucine (L) or a valine (V); (b) SX₂YX₃SLX₄SX₅ (SEQ IDNO: 18), wherein X₂ and X₅ are either a valine or a leucine, wherein X₃is proline (P), serine (S), threonine (T), leucine (L), or valine (V)and wherein X₄ is tryptophan (W), valine (V), leucine (L), isoleucine(I), serine (S) or threorine (T); (c) WLLX₆GTSTX₇ (SEQ ID NO: 19),wherein X₆ is serine (S), threonine (T), isoleucine (I), valine (V) andwherein X₇ is leucine (L) or valine; (d) X₈LIASTTPV (SEQ ID NO: 20,wherein X₈ is one of tyrosine (Y) or tryptophan (W); (e) X₉LIASX₁₀TPV(SEQ ID NO: 21), wherein X₉ is an arginine (R), tyrosine (Y) ortryptophan (W) and X₁₀ is a valine (V), lysine (L), isoleucine (I),serine (S) or threonine (T); or (f) ALYSFLLDFV (SEQ ID NO: 22).